CN113166747B - Protease variants and uses thereof - Google Patents

Protease variants and uses thereof

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Publication number
CN113166747B
CN113166747B CN201980082498.1A CN201980082498A CN113166747B CN 113166747 B CN113166747 B CN 113166747B CN 201980082498 A CN201980082498 A CN 201980082498A CN 113166747 B CN113166747 B CN 113166747B
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seq
protease
gly
serine protease
amino acid
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CN113166747A (en
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昂德里克·赫尔穆特
卡里·云图宁
玛尔加·帕洛赫摩
莱纳·瓦尔塔卡里
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AB Enzymes Oy
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AB Enzymes Oy
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Abstract

The present invention relates to variants of fungal serine proteases having serine protease activity of a protease of the genus arum. Also disclosed are isolated nucleic acid molecules comprising a polynucleotide sequence encoding a fungal serine protease variant, a nucleic acid sequence encoding the protease variant. Host cells and methods of producing polypeptides having serine protease activity are also disclosed. The protease variants may be used as a detergent composition and enzyme preparation for any application in the treatment of fibres, wool, hair, leather or silk, in the treatment of food or feed, or in the modification, degradation or removal of proteinaceous materials.

Description

Protease variants and uses thereof
Technical Field
The present invention relates to variants of serine proteases, and in particular to variants of fungal serine proteases useful in a variety of applications, particularly detergents. The invention further relates to nucleic acid molecules encoding the enzyme variants, recombinant vectors, host cells for producing the enzyme variants, enzyme compositions comprising the enzyme variants and methods of preparing such compositions. The invention also relates to various uses of the enzyme variants and compositions comprising the enzyme variants.
Background
Microbial proteases are one of the most important hydrolases and can be used in various industrial fields such as detergents, foods, leather, pharmaceuticals, diagnostics, waste management and silver recovery. Microbial extracellular proteases account for a major part of the total sales of industrial enzymes worldwide (chemry and Fidantsef, 2003). About 90% of commercial proteases are detergent enzymes (Gupta et al, 2002). Commercial detergent formulations currently in use comprise naturally occurring alkaline serine proteases (EC 3.4.21) derived from the subtilisin family or subtilisin (EC 3.4.21.62) of Bacillus species, or are recombinant protease formulations thereof (Maurer, 2004).
Examples of commercial proteases are e.g. subtilisin CarlsbergSubtilisin 309Subtilisin 147 Cold washing protease(Novozymes A/S,DK)、Ox、Prime(Genencor int., inc., usa), and BLAP S and X series (germany, hangao).
The main problems with the use of proteases in liquid detergents are their instability and proteolytic activity towards other enzymes in the detergent. In liquid detergents, the enzyme is in direct contact with water and chaotropic agents (e.g., anionic surfactants and complexing agents), which can lead to irreversible denaturation. Protease-degrading proteins include themselves and other enzymes in detergent formulations. Surfactants and heat enhance autoproteolysis. Thus, the stability of enzymes in liquid detergents containing proteases represents a major challenge for product development (Maurer, 2010). Various approaches have been used to improve the stability of industrial serine proteases. Based on information derived from comparisons of crystal structure and sequence similarity between homologous proteins, variants with improved stability and/or improved performance can be designed. Variants of natural serine proteases have been developed with improved catalytic efficiency and/or better stability to temperature, oxidants and different wash conditions as well as improved storage stability in liquid detergents by site-directed and/or random mutagenesis. Proteases are deliberately or randomly modified by methods known from the prior art and are optimized for use in, for example, detergents and cleaners. These include site-directed mutagenesis, deletion or insertion mutagenesis, or fusion with other proteins or protein portions. Optimized variants of some proteases are known in the art. Optimized variants are in particular known variants of subtilisin. For example, application WO199523221 describes, among other mutants, mutations in the subtilisin of Bacillus lentus DSM 5483, applications WO2011032988, WO2011141358, WO2012080201 and WO2012080202 describe subtilases based on Bacillus DSM 5483, which, when used, improve protease performance and reduce damage to proteases and other molecules.
Thermophilic mycomycin (Thermomycolin) EC 3.4.21.65, isolated as an extracellular alkaline endopeptidase, is produced by the thermomyces variant sulfurea of the genus Malbranchea pulcella. Thermophilic mycocins are described as 325 residue single chain proteins. It has the active site sequence-Leu-Ser- (Gly) -Thr-Ser x-Met-, which is a typical feature of members of the subtilisin family. Thermophilic mycocins have a disulfide bond, which is very specific. Thermophilic mycotoxins are less thermostable than the extracellular serine proteases of thermophilic bacteria, but are more stable than most fungal proteases (Gaucher and Stevenson, 2004). According to Ong and Gaucher (1975), thermoinactivation of thermophilic mycotoxins occurs in the presence of 10mM Ca 2+ at 73 ℃. Thermophilic mycocins hydrolyze casein over a wide pH range. The optimal pH for casein hydrolysis is about 8.5.EP2691520B1 (AB Enzymes Oy) discloses a thermophilic mycoprotein protease of Oorschot de Hoog ALKO4122 from cladosporium camphora (Malbranchea cinnamomea) (lib.) having improved stability, improved wash performance and stain removal performance compared to the commercial protease products Savinase 16L and Savinase Ultra 16L.
Although numerous patent publications, reviews and articles have been published disclosing serine proteases from various microorganisms, there is still a great need for new proteases which are suitable and effective for modifying, degrading and removing proteinaceous materials of different stains and which are stable in the presence of detergents having highly diverse properties. Stability during storage is also very important due to the autocatalytic nature of serine proteases. However, it is important that other enzymes in the detergent are also stable in the presence of proteases. Protease inhibitors can potentially be used to stabilize proteases in compositions comprising serine proteases. However, inhibitors do not always function optimally, and therefore, there is a particular need for new proteases that are effective in stain removal, but are less aggressive to themselves and other enzymes.
It is also desirable that serine proteases can be produced in large quantities and that downstream processing be performed economically and efficiently by simple separation from cells or mycelium.
Disclosure of Invention
It is an object of the present invention to provide novel protease variants of the genus Pythium (Malbranchea) that are active over a broad pH range and function well over a broad temperature range. Serine proteases for detergent applications must also be stable in the presence of detergents and compatible with detergents. It is a further object of the invention to provide nucleic acid molecules encoding the enzyme variants, recombinant vectors, host cells for producing the enzyme variants, compositions comprising the enzyme variants, methods of preparing such compositions and uses of the enzyme variants and compositions comprising the enzyme variants.
It is an object of the present invention to provide novel variants of a class of arum proteases which are less aggressive towards other enzymes when stored or used in combination with these variants and which also have better detergency properties than the wild-type class of arum proteases, while the stability of the enzyme variants is as good as the stability of the wild-type class of arum proteases.
The fungal serine proteases according to the invention can be produced in high yielding fungal hosts and in processes downstream thereof, e.g. separation of fermentation broths and mycelia is easy to carry out.
The present invention relates to a fungal serine protease variant having serine protease activity and comprising an amino acid sequence having at least 70% identity to the amino acid sequence defined in SEQ ID NO. 2 and comprising amino acid D at position 103 and/or amino acid E at position 105 in the numbering according to SEQ ID NO. 2. The preferred serine protease is a mutant serine protease that is a derivative of the wild-type protease of the species cladosporium pinnatifidum (Malbranchea cinnamomea) (lib.) Oorschot de Hoog.
The inventors found that the same fungal serine protease variants as recombinant serine proteases from the genus arum are herein shown to have better stain removal performance as measured as Δl for different stains under different test conditions than the performance of the wild-type arum protease.
It is an object of the present invention to provide a recombinant serine protease comprising an amino acid sequence having at least 70% identity with the amino acid sequence defined in SEQ ID NO. 2 and which comprises amino acid D at position 103 and/or amino acid E at position 105 in the numbering according to SEQ ID NO. 2.
It is another object of the present invention to provide an isolated nucleic acid molecule comprising a polynucleotide sequence encoding a serine protease selected from the group consisting of:
(a) A nucleic acid molecule encoding a polypeptide having serine protease activity and comprising an amino acid sequence as depicted in SEQ ID NO. 4 or SEQ ID NO. 6 or SEQ ID NO. 8; and
(B) A nucleic acid molecule comprising a coding sequence of a nucleotide sequence as depicted in SEQ ID NO. 3 or SEQ ID NO. 5 or SEQ ID NO. 7.
It is a further object of the present invention to provide a recombinant expression vector comprising an isolated nucleic acid molecule operably linked to regulatory sequences capable of directing the expression and secretion of said serine protease in a suitable host.
Also disclosed are host cells comprising the recombinant expression vectors and enzyme preparations comprising the recombinant serine proteases of the invention.
It is an object of the present invention to provide a method for producing a protease, comprising the steps of: amino acid substitutions Q103D and/or S105E are introduced into a sequence comprising an amino acid sequence having at least 70% identity to the amino acid sequence defined in SEQ ID No. 2.
The use of the recombinant serine protease or enzyme preparation according to the invention in applications for detergents, for treating fibers, for treating proteinaceous material, for treating food or feed or for modification, degradation or removal of proteinaceous material is also an aspect of the invention.
According to the invention, recombinant serine proteases or enzyme preparations can be used as detergent additives. According to the invention, the recombinant serine protease or enzyme preparation may be used in a liquid detergent or a solid detergent, preferably in the form of a bar, a homogeneous tablet, a tablet with two or more layers, a bag with one or more compartments, a conventional or compact powder, a granule (granule), a granule (granulate), a paste, a gel, or a conventional, compact or concentrated liquid. In one aspect the invention also provides a detergent composition comprising a surfactant, a recombinant serine protease or enzyme preparation according to the invention, or an additive selected from the group consisting of stabilizers, buffers, builders, bleaching agents, media, corrosion inhibitors, anti-redeposition agents, corrosion inhibitors, abrasives, optical brighteners, dyes, perfumes, pigments and preservatives.
Brief Description of Drawings
FIG. 1 shows the nucleotide sequence of the Butyrosporum ALK04122 protease gene, its partial promoter (50 nucleotides upstream of ATG) and terminator sequence (60 nucleotides downstream of stop codon) and the deduced amino acid sequence of the encoded protease. Putative signal peptides analyzed by SignalP V3.0 program are indicated in lower case letters and underlined. The pro sequence and the deduced amino acids of the pro sequence are indicated in lowercase letters. Mature nucleotide and peptide sequences are indicated in uppercase letters. Three putative intron sequences are shown in lower case italics and are marked by dashed lines below the nucleotide sequences. The stop codon is shown by an asterisk under the sequence. The mutated amino acids Q103 and S105 are bolded.
FIG. 2 schematically shows the expression cassette pALK452l for producing M59 arum protease variants in Trichoderma reesei (Trichoderma reesei). The 7234bp expression cassette was cleaved from the plasmid vector backbone using NotI restriction enzyme. pcbhl, cbhl promoter; tcbhl, cbhl terminator; s_amdS, a synthetic gene encoding Aspergillus nidulans amidase (Aspergillus nidulans asetamidase) (selectable marker); m59, variant verticillium protease gene. Mutations in the mature amino acid sequence of the variant protease are shown below the name of the variant gene as compared to the wild-type mature sequence. The synthetic amdS gene is expressed from the native Aspergillus nidulans amdS (A. Nidulans amdS) promoter. Expression cassettes pALK4522 and pALK4523 for producing M60 and M61 variants, respectively, are similar in structure to pALK452l for producing M59, as shown.
Fig. 3 schematically shows the detergency performance of the M0 variant compared to the parent molecule M0 in terms of Δl sum of 8 stains. Washing conditions in a reader-Ometer: 40℃l6℃dh,60min, 5g/l commercial liquid detergent, pH approximately 8.2.
Fig. 4 shows the decontamination effect of the M0 variant with the parent molecule M0 in Δl of blood-milk-ink stains at a dose of 6.5BPU per ml of wash. Washing conditions in a reader-Ometer: 40℃l6℃dh,60min, 5g/l commercial liquid detergent, pH approximately 8.2.
Fig. 5 shows the decontamination effect of M0 variant and parent molecule M0 in terms of Δl of egg stains at a dose of 6.5BPU per ml of wash solution. Washing conditions in a reader-Ometer: 40℃l6℃dh,60min, 5g/l commercial liquid detergent, pH approximately 8.2.
Fig. 6 shows the detergency performance of production-like samples of variant M60 compared to the parent molecule M0, in terms of AL sum of 8 stains. Washing conditions in a reader-Ometer: 40℃l6℃dh,60min, 5g/l commercial liquid detergent, pH approximately 8.2.
FIG. 7A shows the decontamination effect of a production-like sample of the M0 variant with the parent molecule M0 in the form of ΔL, CO (E-116) of a blood-milk-ink. Washing conditions in a reader-Ometer: 40℃l6℃dh,60min, 5g/l commercial liquid detergent, pH approximately 8.2.
FIG. 7B shows the decontamination effect of a production-like sample of the M0 variant with the parent molecule M0 in the form of ΔL of blood-milk-ink, CO+PES (E-117). Washing conditions in a reader-Ometer: 40℃l6℃dh,60 minutes, commercial liquid detergent 5g/l, pH approximately 8.2.
Fig. 7C shows the decontamination effect of production-like samples of the M0 variant with the parent molecule M0 in the form of Δl-CO (C-S-37) of pigmented whole egg stains. Washing conditions in a reader-Ometer: 40℃l6℃dh,60 minutes, commercial liquid detergent 5g/l, pH approximately 8.2.
FIG. 7D shows the decontamination effect of a production-like sample of the M0 variant with the parent molecule M0 in the form of ΔL, CO (C-S-38) of pigmented yolk eggs. Washing conditions in a reader-Ometer: 40℃l6℃dh,60 minutes, commercial liquid detergent 5g/l, pH approximately 8.2.
FIG. 8 depicts the degradation effect of the M0 variant (culture sample) on commercial cellulase (1%) in liquid detergent at 37℃and 7 days using protease amounts corresponding to 520BPU/g activity. The stability of the cellulase after storage is shown as relative residual activity compared to the solution containing the parent molecule M0 as protease.
FIG. 9 depicts the degradation effects of a production-like sample of variant M60 with M0 cellulase (1%) in a liquid detergent at 37℃and 7 days, using a high protease amount corresponding to 2080BPU/g activity. The stability of the cellulase is shown as relative residual activity after storage compared to a solution containing the parent molecule M0 as protease.
Sequence listing
SEQ ID NO:1 encoding an amino acid sequence of an mature arum ALK04122 protease. The full-length gene is contained in plasmid pALK3094 as described in EP2691520B 1.
SEQ ID NO:2 mature arum ALK04122 protease, comprising amino acids Alal l to Arg40l of protease.
SEQ ID NO:3 encoding the amino acid sequence of the mature arum ALK04122 protease, having the mutation CAG- > GAC or CAG- > GAT at positions 307-309, resulting in the mutation Q103D in the mature protease sequence.
SEQ ID NO:4 the amino acid sequence of an M59 derivative of the mature verticillium ALK04122 protease, comprising amino acids Alal l to Arg40l of the mature protease and mutation Q103D at position 103 of the mature protease.
SEQ ID NO:5 encoding the amino acid sequence of the mature arum ALK04122 protease, has a mutation at positions 313-315 of TCA- > GAG or TCA- > GAA, resulting in a mutation in the mature protease sequence S105E.
SEQ ID NO: the amino acid sequence of the M60 derivative of the mature verticillium ALK04122 protease, including amino acids Alal l to Arg40l of the protease and mutation S105E at position 105 of the mature protease.
SEQ ID NO:7 the nucleotide sequence encoding the amino acid sequence of the mature arum ALK04122 protease has mutations CAG- > GAC and TCA- > GAT at positions 307-309 and 313-315, respectively.
SEQ ID NO: the amino acid sequence of the M61 derivative of the mature arum ALK04122 protease, including amino acids Alal l to Arg40l of the protease and mutations Q103D and S105E at positions 103 and 105, respectively, of the mature protease.
SEQ ID NO:9 a nucleotide sequence encoding the amino acid sequence of the full length verticillium ALK04122 protease.
SEQ ID NO: amino acid sequence (including signal sequence, pro sequence and mature form sequence) of the 10 full length verticillium ALK04122 protease.
Detailed Description
The present invention provides novel serine protease variants of fungal origin.
Herein, "derived from" refers not only to serine proteases produced or producible by the strain of the organism in question, but also to serine proteases encoded by DNA sequences isolated from such strains and produced in host organisms transformed with said DNA sequences. Finally, the term is intended to mean a serine protease which is encoded by a DNA sequence of synthetic and/or cDNA origin and has the recognition characteristics of the serine protease in question.
The fungal serine protease variants of the invention are based on a protease of the genus arum, preferably a protease based on the genus arum (lib.) Oorschot de Hoog (synonym for the Malbranchea pulchella variant sulfurea (Miehe) Cooney & r.mers). The full-length fungal serine protease enzyme on which the variants of the invention are based are encoded by the polynucleotide sequence contained in pALK3094, pALK3094 being deposited in e.coli (ESCHERICHIA COLI) RF8791 under accession No. DSM 24410 as disclosed in patent EP 2691520B 1 (AB enzyme Oy).
Preferably, the present invention relates to a fungal serine protease variant having serine protease activity and comprising the amino acid sequence of a mature protease as defined in SEQ ID NO. 4 (M59) or the amino acid sequence of a mature protease as defined in SEQ ID NO. 6 (M60) or the amino acid sequence of a mature protease as defined in SEQ ID NO. 8 (M61).
The fungal serine protease variants of the invention are produced from a recombinant expression vector comprising a nucleic acid molecule encoding a fungal serine protease variant of the invention operably linked to regulatory sequences capable of directing the expression of the serine protease in a suitable host. Non-limiting examples of host cells are fungal cells, filamentous fungal cells from ascomycota (Ascomycota), discomycotina (Pezizomycotina); preferably selected from the group consisting of Hymenochaetaceae (Sordariomycetes), hymenochaetaceae (Hypocreomycetidae), hymenochaetales (Hypocreales) and Botrytis (Microascales), aspergillus (Aspergillus), chrysosporium (Chrysosporium), myceliophthora (Myceliophthora) and Humicola; More preferably selected from the group consisting of Hymenochaetaceae (Sordariomycetes), hymenochaetaceae (Hypocreomycetidae), hymenochaetales (Hypocreales) and Botrytis (Microascales), aspergillus (Aspergillus), chrysosporium (Chrysosporium), myceliophthora (Myceliophthora) and Humicola; More preferably selected from the group consisting of Hypocreaceae (Hypocreacea), eichhornidae (NECTRIACEAE), clavipitaceae (CLAVICIPITACEAE), microcystaceae (Microascaceae) and Trichoderma (Trichoderma) (asexual Hypocrea, anamorph of Hypocrea), fusarium (Fusarium), gibberella (Gibberella), saccharum (Nectria), stachybotrys (Stachybotrys), clavipitaceae (CLAVICEPS), Metarhizium (Metarhizium), ustilago (Villosiclava), nematoda (Ophiocordyceps), cephalosporanium (Cephalosporium) and podophyllum (Scedosporium); more preferably selected from Trichoderma reesei (Trichoderma reesei) (Oenothera roseum, hypocrea jecorina), trichoderma citrinoviride (T.citrinoviridae), trichoderma longibrachiatum (T.virens), trichoderma viride (T.virens), trichoderma harzianum (T.harzianum), trichoderma asperellum (T.asprellum), trichoderma atroviride (T.atroviridae), trichoderma parapsilosis (T.parameresei), fusarium oxysporum (Fusarium oxysporum), Fusarium graminearum (F. Graminearum), fusarium pseudograminearum (F. Pseudoplastic), fusarium venenatum (F. Venenatum), gibberella vinifera (Gibberella fujikuroi), gibberella moniliformis (G. Moniliformis), gibberella zeae (G. Zeaea), xanthomonas comucocalyxa (Nectria haematococca) (Saccharum, haematonectria), stachybotrys (Stachybotrys chartarum), Acremonium (S.chlorohalonata), clavipita (CLAVICEPS PURPUREA) ryegrass (CLAVICEPS PURPUREA), metarhizium anisopliae (Metarhizium acridum), metarhizium anisopliae (M.anicoplia), rhizoctonia cerealis (Villosiclava virens), cordyceps (Ophiocordyceps sinensis), cephalosporium acremonium (Acremonium (Cephalosporium) chrysogenum) and Actinobacillus tip (Scedosporium apiospermum), Aspergillus niger (Aspergillus niger), aspergillus awamori (Aspergillus awamori), aspergillus oryzae (Aspergillus oryzae), chrysosporium rupestis (Chrysosporium lucknowense), myceliophthora thermophila (Myceliohpthora thermophila), humicola insolens (Humicola insolens) and Humicola grisea; Most preferred is Trichoderma reesei. Non-limiting examples of host cells are yeast (e.g., saccharomyces cerevisiae (Saccharomyces cerevisiae), pichia pastoris, yarrowia lipolytica (Yarrowia lipolytica)) and bacterial cells, preferably gram positive bacilli (e.g., bacillus subtilis (Bacillus subtilis), bacillus licheniformis (B.lichenifermis), bacillus megaterium (B.megaterium), bacillus amyloliquefaciens (B.amyloliquefaciens), bacillus pumilus), gram-negative bacteria (e.g., E.coli (ESCHERICHIA COLI)), and actinomycetes (e.g., streptomyces sp.).
In one embodiment, the host cell is a fungal cell, preferably a filamentous fungal cell, such as Aspergillus, trichoderma or Trichoderma reesei. In one embodiment, the host cell is a bacterial cell, preferably a gram positive bacillus cell, such as bacillus subtilis, bacillus licheniformis, bacillus megaterium, bacillus amyloliquefaciens, or bacillus pumilus.
Preferably, the enzyme variant is produced in trichoderma or aspergillus, most preferably trichoderma reesei.
The invention also relates to an isolated nucleic acid molecule comprising a polynucleotide sequence encoding a serine protease variant selected from the group consisting of:
(a) A nucleic acid molecule encoding a polypeptide having serine protease activity and comprising an amino acid sequence as depicted in SEQ ID NO. 4;
(b) A nucleic acid molecule encoding a polypeptide having serine protease activity and comprising an amino acid sequence as depicted in SEQ ID NO. 6;
(c) A nucleic acid molecule encoding a polypeptide having serine protease activity and comprising an amino acid sequence as depicted in SEQ ID NO. 8;
(d) A nucleic acid molecule comprising a coding sequence for a nucleotide sequence as depicted in SEQ ID NO. 3;
(d) A nucleic acid molecule comprising a coding sequence for a nucleotide sequence as depicted in SEQ ID NO. 5; and
(D) A nucleic acid molecule comprising a coding sequence of a nucleotide sequence as depicted in SEQ ID No. 7.
The present invention relates to a method for producing a polypeptide having serine protease activity, said method comprising the steps of culturing a host cell of the invention and recovering the polypeptide. In addition, polypeptides having serine protease activity, which are encoded by the nucleic acid sequences of the invention and which can be obtained by the above-described methods, are also within the scope of the invention.
The present invention relates to a method for obtaining an enzyme preparation comprising the steps of culturing a host cell according to the invention and recovering the polypeptide from the cell or isolating the cell from the culture medium and obtaining a supernatant. Enzyme preparations obtainable by the above-described methods are also within the scope of the invention.
The present invention relates to an enzyme preparation comprising a serine protease variant or a plurality of serine protease variants of the invention.
The term "mutant" or "variant" is also used herein to refer to serine proteases that contain mutations relative to the corresponding wild-type serine protease.
The invention further relates to compositions comprising a serine protease variant or serine protease variants of the invention.
The enzyme preparation or composition (e.g. detergent preparation) comprising the protease variant or variants of the invention may further comprise a suitable additive selected from proteases (other proteases than the proteases of the invention), amylases, cellulases, lipases, xylanases, mannanases, cutinases, pectinases, polygalacturonases, pectate lyases, pectolyases, esterases, phytases, arabinases, galactanases, huang Yuanmei, xyloglucanases, dnases, laccases and/or peroxidases and oxidases with or without a mediator, and selected from stabilizers, buffers, surfactants, bleaches, mediators, corrosion inhibitors, builders, anti-redeposition agents, optical brighteners, dyes, pigments, perfumes, corrosives, abrasives, and preservatives, etc.
The used medium of the production host may be used for this (used as culture), or the host cells may be removed and/or concentrated, filtered or fractionated. Or sun drying. Enzyme preparations and compositions comprising serine proteases of the invention may be in the form of liquid compositions or solid compositions, such as solutions, dispersions, pastes, powders, granules, granulates, coated granulates, tablets, cakes, crystals, crystal slurries, gels or pills. The enzyme may be in an immobilized form in the formulation or composition.
The invention also includes the use of a serine protease variant or serine protease variants or enzyme preparations of the invention for detergents, for treating fibers, for treating proteinaceous material (e.g. wool, hair, leather, silk), for treating food or feed, or for any application involving modification, degradation or removal of proteinaceous material. The enzyme composition is used in the textile and detergent industry, biomass processing and biomass hydrolysis, preferably in the biofuel, starch, pulp and paper industry, food, baking, feed or beverage industry. In particular, the enzyme or enzyme preparation may be used as a detergent additive in a liquid detergent or a solid detergent, preferably as a bar, a homogeneous tablet, a tablet with two or more layers, a bag with one or more compartments, a conventional or compact powder, granules, paste, gel, or a conventional, compact or concentrated liquid.
These enzyme variants are active in washing over a wide pH and temperature range and perform particularly well over low temperatures as well as at medium and high temperatures. Enzyme variants are ideal choices for detergent applications, are tolerant of typical detergent compositions, and are effective at low enzyme levels in detergent solutions. In particular, the protease variants are active at application temperatures of from 0 ℃ to 90 ℃, preferably ranging from 5 ℃ to 60 ℃. Each fungal serine protease variant of the invention is capable of degrading or removing proteinaceous stains in the presence of a detergent at a temperature of from 0 ℃ to 90 ℃, preferably at a temperature of from 5 ℃ to 60 ℃. The fungal serine protease variants of the invention are particularly suitable for effectiveness at temperatures equal to or below 60 ℃ depending on the washing conditions and the adjunct ingredients and additives in the detergent.
The proteases of the invention are also highly stable in liquid detergent compositions. Accordingly, the present invention provides novel serine protease variants for use in detergents and other applications, particularly in liquid formulations. The fungal serine protease variants may be produced in a high yielding fungal host and downstream processing thereof, e.g. separation of fermentation broth and mycelium is facilitated.
"Serine protease" or "serine endopeptidase" or "serine endoprotease" in connection with the present invention refers to an enzyme classified as EC 3.4.21 by the international union nomenclature of biochemistry and molecular biology. Proteases can be classified using group-specific inhibitors. Different groups of serine protease inhibitors include synthetic chemical inhibitors and natural protein inhibitors. Thus, serine protease activity can be determined in assays based on cleavage of a specific substrate or in assays using any protein-containing substrate containing a substrate with or without a specific inhibitor of serine protease under suitable conditions.
The term "serine protease activity" as used herein refers to hydrolytic activity towards protein containing substrates such as casein, hemoglobin and BSA. Methods for the analysis of proteolytic activity are well known in the literature, for example as mentioned in Gupta et al (2002).
Serine proteases are synthesized as inactive proenzyme precursors or pro-coenzymes in the form of proenzymes that are activated by removal of the signal sequence (secretion signal peptide or propeptide) and the pro-sequence (propeptide) to produce the active mature form of the enzyme (Chen and Inouye, 2008).
Examples of suitable signal sequences are those of fungal or yeast organisms. Such signal sequences are well known in the literature. Suitable pro-sequences are those of fungal or yeast or bacterial proteases.
Recombinant vectors may also contain sequences that facilitate integration of the vector into the host chromosomal DNA for stable expression and/or to facilitate targeting to specific locations in the host genome.
The term "mature" means that the serine protease form from which the signal sequence (propeptide) has been removed and that the propeptide comprises enzymatically or catalytically active essential amino acids. In filamentous fungi, it is the natural form secreted into the culture medium. The first amino acid of the mature sequence can be determined by N-terminal sequencing of the secreted protease. If no biochemical data is available, the N-terminal position can be estimated by aligning the amino acid sequence with the mature amino acid sequence of the homologous protein. Alignment can be performed using, for example, a ClustalW2 alignment (http:// www.ebi.ac.uk/Tools/msa/ClustalW2 /).
In order to improve the performance of the arum serine protease in various industrial applications, for example in detergents, it is desirable to improve the properties of the native enzyme. These properties include, for example, storage stability, stability in the presence or absence of detergents, pH stability, oxidation stability or resistance to bleaching agents and substrate specificity. The autoproteolytic activity of the enzyme has an influence on the storage stability and should be as low as possible. It is self-evident that the wash performance of the modified protease should not be impaired compared to the parent or precursor protease, for example in laundry and dish wash compositions. In other words, enzyme variants are expected to have similar or even improved wash performance and stain removal properties compared to the parent serine protease.
The serine protease variants produced can be purified by conventional methods using enzyme chemistry, such as salt preparation, ultrafiltration, ion exchange chromatography, affinity chromatography, gel filtration and hydrophobic interaction chromatography. Purification can be monitored by protein assay, enzyme activity assay and SDS polyacrylamide gel electrophoresis. The enzyme activity and stability of the purified enzyme at various temperatures and pH values, and the molecular weight and isoelectric point can be determined.
Protease activity is generally based on degradation of soluble substrates. In detergent applications, proteases must act on at least partially insoluble materials. Thus, an important parameter of detergent proteases is the ability to adsorb to and hydrolyze these insoluble fragments.
The fungal serine protease variants of the invention function at the temperatures defined above in the presence of detergents, in particular, the fungal serine protease variants have good properties in the presence of detergents. Fungal serine protease variants from the genus arum have better stain removal performance, measured as Δl, than wild-type arum protease under different test conditions for different stains.
According to a preferred embodiment of the invention, each of the recombinant fungal serine protease variants is a polypeptide having serine protease activity comprising an amino acid sequence which is at least 70% identical to the amino acid sequence of the mature arum alk o4122 protease defined in SEQ ID No. 2 and comprising an amino acid substitution at position 103 or position 105 or positions 103 and 105. According to a more preferred embodiment, the substitution at position 103 is Q103D and the substitution at position 105 is S105E. According to a more preferred embodiment, the substitution at position 103 is Q103D and the substitution at position 105 is S105E.
According to a preferred embodiment of the invention, the recombinant serine protease comprises an amino acid sequence having at least 70% identity with the amino acid sequence defined in SEQ ID NO. 2 and comprises amino acid D at position 103 and/or amino acid E at position 105 in the numbering according to SEQ ID NO. 2. Preferably, the recombinant serine protease has improved detergency.
According to a preferred embodiment of the invention, the recombinant serine protease comprises an amino acid sequence having at least 70% identity with the amino acid sequence defined in SEQ ID NO. 2 and comprises amino acid D at position 103 in the numbering according to SEQ ID NO. 2 and has improved detergency properties.
According to a preferred embodiment of the invention, the recombinant serine protease comprises an amino acid sequence having at least 70% identity with the amino acid sequence defined in SEQ ID NO. 2 and comprises amino acid E at position 105 in the numbering according to SEQ ID NO. 2 and has improved detergency properties.
Preferably, the protease variant polypeptide comprises an amino acid sequence having at least about 70%, and more preferably up to at least about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 90.5%, about 91%, about 91.5%, about 92%, about 92.5%, about 93%, about 93.5%, about 94%, about 94.5%, about 95%, about 95.5%, about 96%, about 96.5%, about 97%, about 97.5%, about 98%, about 98.5% and about 99% identity to the amino acid sequence of the mature Butyrosporum ALK04122 protease defined in SEQ ID NO. 2.
According to a preferred embodiment of the invention, the recombinant serine protease comprises an amino acid sequence having at least 82% identity to the amino acid sequence defined in SEQ ID NO. 2 and comprises amino acid D at position 103 and/or amino acid E at position 105 in the numbering according to SEQ ID NO. 2. Preferably, the recombinant serine protease has improved detergency.
According to a preferred embodiment of the invention, the recombinant serine protease comprises an amino acid sequence having at least 82% identity to the amino acid sequence defined in SEQ ID NO. 2 and comprises amino acid D at position 103 in the numbering according to SEQ ID NO. 2 and has improved decontamination properties.
According to a preferred embodiment of the invention, the recombinant serine protease comprises an amino acid sequence having at least 82% identity to the amino acid sequence defined in SEQ ID NO. 2 and comprises amino acid E at position 105 in the numbering according to SEQ ID NO. 2 and has improved decontamination properties.
The term "identity" refers herein to the identity between two amino acid sequences compared to each other within a corresponding sequence region having about the same amount of amino acids. For example, the identity of the mature sequences of two amino acid sequences can be compared. The amino acid sequences of the two molecules to be compared may differ in one or more positions, but this does not alter the biological function or structure of the molecules. Such variations may occur naturally in different organisms, or due to mutations in the amino acid sequence, or they may be achieved by specific mutagenesis. Variations may be caused by deletions, substitutions or insertions at one or more positions in the amino acid sequence. Sequence identity was measured by using ClustalW2 alignment (http:// www.ebi.ac.uk/Tools/msa/ClustalW2 /) with default settings (protein weight matrix: gonnet, gap open: 10, gap extension: 0.20, gap distance 5).
Serine proteases, suitably fungal serine proteases of the invention have "good performance in the presence of detergents", i.e. the ability to degrade or remove proteinaceous stains or materials in the presence of detergents over a broad temperature range, in particular over a temperature range below that of currently commercial subtilisin products. The enzymes of the invention function well between 5℃and 60℃in the presence of detergents.
A preferred embodiment of the invention is a fungal serine protease enzyme encoded by an isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 3. SEQ ID NO:5 or SEQ ID NO:7.
One embodiment of the invention is a serine protease produced by a recombinant expression vector comprising a nucleic acid molecule encoding a fungal serine protease variant as characterized above, operably linked to regulatory sequences capable of directing the expression and secretion of said serine protease in a suitable host, as described above. Construction of the recombinant expression vector and use of the vector is described in more detail in EP2691520B 1.
The invention also relates to an isolated nucleic acid molecule comprising a polynucleotide sequence encoding a serine protease selected from the group consisting of:
(a) A nucleic acid molecule encoding a polypeptide having serine protease activity and comprising an amino acid sequence as depicted in SEQ ID NO. 4, SEQ ID NO. 6 or SEQ ID NO. 8; and
(B) A nucleotide sequence encoding the amino acid sequence of a mature arum ALK04122 protease as depicted in SEQ ID No. 3, SEQ ID No. 5 or SEQ ID No. 7 with the mutations CAG- > GAC or GAT and/or TCA- > GAG or GAA (at positions 307-309 and/or positions 313-315).
The nucleic acid molecule of the invention may be DNA or RNA, wherein the DNA may constitute genomic DNA or cDNA or synthetic DNA. RNA may constitute natural RNA or synthetic RNA.
Thus, an isolated polynucleotide sequence or an isolated nucleic acid molecule is within the scope of the invention that encodes a fungal serine protease variant or polypeptide comprising the amino acid sequence of SEQ ID NO:2, and the mature form of the amino acid sequence of the arum ALK04122 enzyme described in 2.
The invention also relates to recombinant expression vectors or recombinant expression constructs useful for propagating or expressing nucleic acid sequences encoding selected serine protease variants in suitable prokaryotic or eukaryotic hosts. Recombinant expression vectors comprise DNA or nucleic acid sequences that facilitate or direct the expression and secretion of a serine protease encoding sequence in a suitable host, such as promoters, enhancers, terminators (including transcription and transfer termination signals), and signal and prosequences operably linked to polynucleotide sequences encoding the serine protease. The expression vector may further comprise a marker gene for selection of transformants, or the selectable marker may be introduced into a host in another vector construction by co-transformation. The regulatory sequences may be homologous or heterologous to the producing organism, or they may originate from the organism from which the gene encoding the serine protease was isolated.
Examples of promoters for expressing serine proteases of the invention in filamentous fungal hosts are promoters of the natural proteins of Trichoderma reesei early summer rains, such as cellobiohydrolase 1, aspergillus oryzae TAKA amylase, alkaline protease ALP and triose phosphate isomerase, rhizopus niveus (Rhizopus miehei) lipase, aspergillus niger or Aspergillus awamori glucoamylase (glaA), fusarium oxysporum trypsin-like protease, and Chrysosporium rufin cellobiohydrolase 1 promoters.
In yeast, promoters such as Saccharomyces cerevisiae enolase (ENO-1), galactokinase (GAL 1), alcohol dehydrogenase (ADH 2), and 3-phosphoglycerate kinase may be used to provide expression.
Examples of promoter sequences for directing transcription of serine proteases of the invention in bacterial hosts are the promoter of the lac operon of E.coli, the Streptomyces coelicolor (Streptomyces coelicolor) agarase dag A promoter, the promoter of the Bacillus licheniformis alpha-amylase gene (arnyL), the promoter of the Bacillus stearothermophilus (B.stearothermophilus) maltogenic amylase gene (amyM), the promoters of the Bacillus subtilis xylA and xylB genes, and the like.
Suitable terminators include the terminators of the genes described above or any other characterized terminator sequence.
Suitable transformation or selection markers include those that confer selectable properties on the transformant, such as Aspergillus amdS (Aspergillus amdS) or complement a defect in the host, such as the Aspergillus niaD or dal genes from Bacillus subtilis or Bacillus licheniformis. Selection may also be based on markers conferring antibiotic resistance, such as ampicillin, kanamycin, chloramphenicol, tetracycline, phleomycin or hygromycin resistance (hygromycin resistance).
The invention also relates to a host cell comprising the recombinant expression vector. Suitable hosts for the production of fungal serine proteases are homologous or heterologous hosts, such as microbial hosts, including fungi, yeasts and bacteria. Production systems in plant or mammalian cells are also possible.
Suitable expression and production host systems are, for example, production systems developed for filamentous fungal hosts selected from the group consisting of the classes Hypocreaceae, hypocreaceae and Botrytis, aspergillus, chrysosporium, myceliophthora and Humicola; more preferably, the sarcodaceae, the erythromycetaceae, the ergotaceae, the microcystaceae and the trichoderma (asexual sarcoidosis), the fusarium, the gibberella, the rubus, the viticola, the ergot, the metarhizium, the ustilago, the nematoda, the cephalosporanges and the podophyllum; more preferably selected from Trichoderma reesei (Trichoderma reesei), trichoderma citrinovyi, trichoderma longibrachiatum, trichoderma viride, trichoderma harzianum, trichoderma aspergillum, trichoderma reesei, fusarium oxysporum, fusarium graminearum, fusarium pseudograminearum, fusarium venenatum, gibberella algoides, gibberella fulgidus, gibberella zeae, gibberella flagelliforme (Gibberella), stachybotrytis, stachybotrys, erysiphe seche, metarhizium anisopliae, rhizoctonia solani, cordyceps, cephalosporium acremonium, actinobacillus tenuipes, aspergillus niger, aspergillus awamori, trichosporotrichum, myces thermophilus, humicola and Humicola grisedge; most preferred is Trichoderma reesei. Non-limiting examples of production systems are yeasts (e.g., saccharomyces cerevisiae, pichia pastoris, yarrowia lipolytica) and bacterial cells, preferably gram positive bacilli (e.g., bacillus subtilis, bacillus licheniformis, bacillus megaterium, bacillus amyloliquefaciens, bacillus pumilus), gram negative bacilli (e.g., escherichia coli), and actinomycetes (e.g., streptomyces).
The invention also relates to a method for producing a polypeptide having serine protease activity, comprising culturing a native or recombinant host cell carrying a recombinant expression vector for a serine protease variant of the invention under suitable conditions and optionally isolating the enzyme. The production medium is a medium suitable for culturing host organisms and may contain an inducer for efficient expression. Suitable media are well known in the literature.
The present invention relates to polypeptides having serine protease activity, which are encoded by the nucleic acid molecules of the invention and obtainable by the above-described methods.
The invention further relates to a method for obtaining an enzyme preparation comprising a polypeptide having serine protease activity, said method comprising the steps of culturing a host cell carrying an expression vector of the invention and recovering the polypeptide from the cell or isolating the cell from the culture medium and obtaining a supernatant having serine protease activity.
The invention also relates to an enzyme preparation comprising a serine protease variant or serine protease variants as described above. The enzyme preparation or composition has serine protease activity and is obtainable by the method according to the invention.
Enzyme preparations and compositions comprising a serine protease variant or variants of the invention are also within the scope of the invention.
The enzyme preparation or composition (e.g. detergent preparation) comprising a protease variant or protease variants of the invention may further comprise other enzymes selected from proteases (other proteases than the proteases of the invention), amylases, lipases, cellulases, cutinases, pectinases, polygalacturonases, pectin lyase, esterases, phytases, arabinases, galactanases, huang Yuanmei, xyloglucanases, dnases, mannanases, xylanases and oxidases, e.g. laccase or peroxidases with or without mediator. These enzymes are expected to enhance the performance of the serine proteases of the present invention, for example, by removing carbohydrates and oils or fats present in the material to be treated. The enzyme may be a native or recombinant enzyme produced by the host strain or may be added to the culture supernatant or formulation or composition after the production process.
The enzyme preparation or composition may further comprise one or more suitable additives selected from the group consisting of surfactants, builders, chelating agents (chelators) or chelating agents (CHELATING AGENTS), bleaching systems or bleach components, polymers, fabric conditioning agents, suds boosters, suds suppressors, dyes, perfumes, light brown inhibitors (tannish inhibitor), light brighteners (optical brighteners), bactericides, fungicides, soil suspending agents, preservatives (anticorrosion agents), hydrotropes, fabric hueing agents, dispersants, dye transfer inhibitors, fluorescent brighteners (fluorescence WHITENING AGENTS), soil release polymers (soil release polymers), anti-redeposition agents, anti-shrink agents, anti-wrinkle agents, bactericides, adhesives, carriers, dyes, enzyme stabilizers, fabric softeners, fillers, suds modifiers, perfumes, pigments, buffers, preservatives (PRESERVATIVES), turf inhibitors, structural agents for solvents and liquid detergents, structural elastants, enzyme inhibitors or stabilizers, enzyme activators, transferases, hydrolases, oxidoreductases, bluing agents and fluorescent dyes, antioxidants and solubilizing agents.
Surfactants can be used to emulsify oils and wet surfaces. The surfactant may be nonionic and/or anionic and/or cationic and/or zwitterionic including semi-polar. Buffers may be added to the enzyme preparation or composition to alter the pH or affect the performance or stability of other ingredients. Suitable stabilizers include polyols such as propylene glycol or glycerol, sugars or sugar alcohols, sorbitol or hexylene glycol, lactic acid, boric acid or boric acid derivatives, formic acid, aromatic borates, phenylboronic acid derivatives, peptides, other reversible subtilisin inhibitors, or combinations thereof.
Bleaching agents are used to oxidize and degrade organic compounds. Examples of suitable chemical bleaching systems are sources of H 2O2, such as perborates or percarbonates, bleach activators with or without formation of peroxyacids, such as tetraacetylethylene diamine, or alternative peroxyacids, such as amides, imides or sulfones. Chemical oxidants may be replaced in part or in whole by the use of oxidases such as laccase or peroxidase. Many laccases do not function effectively without mediators.
Builders or complexing agents include zeolites, bisphosphates, triphosphates, carbonates, citrates and the like. The enzyme formulation may also comprise one or more polymers, such as carboxymethyl cellulose, poly (ethylene glycol), poly (vinyl alcohol), poly (vinyl pyrrolidone), and the like. In addition, softeners, corrosives, preservatives to prevent deterioration of other components, abrasives, and substances to alter foaming and viscosity characteristics may also be added.
According to a preferred embodiment of the invention, the enzyme preparation or composition comprising the enzyme variant is in the form of a liquid composition or a solid composition, such as a solution, dispersion, paste, powder, granule, granulate, coated granulate, tablet, cake, crystal slurry, gel or pill. According to a preferred embodiment of the invention, the composition is a liquid detergent or a solid detergent, preferably a bar, a homogeneous tablet, a tablet with two or more layers, a bag with one or more compartments, a conventional compact powder, granules, paste, gel, or a conventional, compact or concentrated liquid. Furthermore, the enzyme variant in the formulation or composition may be in the form of an immobilized enzyme.
Serine proteases of the invention can be used like other proteases, in particular alkaline proteases, in detergents, proteins, brewing, meat, photography, leather, dairy and pharmaceutical industries (Kalisz, 1988; rao et al, 1998). For example, it can be used as a substitute for chemicals that convert fibrous protein waste (e.g., horn, feathers, nails, and hair) into useful biomass, protein concentrate, or amino acids (Anwar and Saleemuddin, 1998). The serine protease of the present invention can be used as other proteases to prove successful in improving leather quality and reducing environmental pollution and saving energy, and it can be used for peptide synthesis and resolution of D, L-amino acid mixtures. The combination of subtilisin with broad-spectrum antibiotics for the treatment of burns and wounds is an example of the use of serine proteases in the pharmaceutical industry, and thus the fungal serine proteases of the present invention can also be considered to have such uses, as well as for the removal of blood from surgical equipment and cleaning of contact lenses or dentures. Like alkaline protease from Phytophthora fly pathogen (Conidiobolus coronatus), the fungal serine protease variants of the invention can be used to replace trypsin in animal cell culture. The proteases of the invention are also useful for cleaning membranes and disrupting biological membranes. In baking, proteases may be used, for example in other food applications in breaking gluten networks and hydrolyzing food proteins, such as proteins in milk. They can also be used, for example, in the treatment of yeasts, in refining (extraction of more proteins from animal bones), in creating new flavors, in reducing bitter taste, in modifying emulsifying properties, in producing bioactive peptides and in reducing protein allergies. Substrates include animal, plant and microbial proteins.
The detergent industry, and in particular the washing powder industry, has become the single major consumer of proteases active in the high pH range (Anwar and Saleemuddin, 1998). Ideal detergent proteases should have a broad substrate specificity to facilitate removal of various stains caused by food, grass, blood and other body secretions. It must be active at the pH and ionic strength of the detergent solution, at the wash temperature and pH, and be able to withstand mechanical handling as well as chelating and oxidizing agents added to the detergent. Because of energy conservation concerns, it is currently desirable to use proteases at lower temperatures.
The invention also relates to the use of serine protease variants or enzyme variant preparations for detergents, for treating textile fibres, for treating proteinaceous materials such as wool, hair, silk, leather, for treating feed or food, or for any application involving modification, degradation or removal of proteinaceous materials.
Thus, a preferred embodiment of the present invention is the use of a serine protease variant as described above as a detergent additive useful in laundry detergents and dishwashing compositions, including automatic dishwashing compositions.
The expression "detergent" is used to denote a substance or material intended to aid cleaning or having cleaning properties. The term "detergency" refers to the presence or degree of cleaning performance. The extent of cleaning performance can be tested on different proteins or protein-containing substrate materials or stains or stain mixtures, which are bound to solid, water insoluble carriers such as textile fibres or glass. Typical proteinaceous materials include blood, milk, ink, eggs, grasses and sauces. For testing purposes, a variety of protein stains are commercially available. The detergent enzymes function to degrade and remove protein-containing stains. The test results depend on the type of stain, the composition of the detergent, and the nature and status of the textiles used in the wash test (Maurer, 2004).
In the present invention, the term "detergent stability" means that the enzyme or enzyme variant substantially retains its activity in the detergent solution during storage and/or washing. Thus, stains or materials may be effectively degraded or removed in the presence of the detergent. Stability can be analyzed by determining residual activity, for example after incubation in detergent for several days (37 ℃). Stability may be measured by specific activity of various enzymes or by application of a test as wash performance.
The term "effective amount" of serine protease refers to the amount of protease necessary to achieve enzymatic activity in a particular detergent composition. Preferably, the detergent composition of the present invention comprises from about 0.0001% to about 10% of the protease variant of the present invention (as an enzyme protein), more preferably from 0.001% to about 1%, still more preferably from 0.001% to about 0.5%, by weight of the detergent composition.
In general, the wash performance of proteases is measured as "stain removal efficiency" or "stain removal effect" or "degree of cleaning performance", meaning a visible and measurable increase in brightness or color change of the stained material, for example, in a artificially soiled sample or test cloth. The change in luminance or color value can be measured, for example, by measuring color as a reflectance value from the L x a x b x color space coordinates using a spectrophotometer. The discoloration or removal of protein stains, which represents the performance (detergency effect or efficiency) of the protease, is for example calculated as Δl, which represents the brightness value L of the enzyme treated fabric minus the brightness value L of the fabric treated with the wash liquor without enzyme (enzyme blank or control). The presence of detergent may enhance the stain removal performance of the enzyme. Serine proteases of the invention degrade various protein stains (cocoa (E-112), blood/milk/ink, co+PES (E-117), blood/milk/ink, co (E-116), grass (E-164), pigmented whole egg (CS-37), pigmented egg yolk (CS-38), chocolate milk soot (C-03) and peanut oil, milk (C-10)) in the presence of detergent under alkaline conditions (example 2).
It is an object of the present invention to provide novel variants of the class of arum proteases which have better detergency properties (e.g. blood-milk-ink, pigment whole egg and pigment egg yolk) than the commercially available enzymes and wild-type class of arum proteases, whereas the stability of the enzyme variants is as good or better than the wild-type class of arum proteases. In addition, these variants of the invention are milder or less aggressive to other enzymes such as cellulases, as shown in FIGS. 8 and 9 of example 3.
In addition to washing, the enzyme variants of the invention substantially retain their activity during storage, even when stored in a liquid detergent, such as the arum ALK04122 protease (data not shown).
According to a preferred embodiment of the invention, the fungal serine protease variants of the invention are useful in detergent liquids and detergent powders. The enzyme variants of the enzyme preparations of the invention may be formulated for hand or machine washing, or may be formulated for hard surface cleaning or hand or machine dishwashing operations.
One aspect of the invention is a recombinant serine protease comprising an amino acid sequence having at least 70% identity to the amino acid sequence defined in SEQ ID NO. 2 and comprising amino acid D at position 103 and/or amino acid E at position 105 in the numbering according to SEQ ID NO. 2. A preferred aspect of the invention is a recombinant serine protease, wherein, in the sequence according to SEQ ID NO:2, said polypeptide having at least one amino acid substitution selected from the group consisting of Q103D and S105E. According to a more preferred aspect of the invention, the amino acid sequence of the recombinant serine protease corresponds to one of the amino acid sequences depicted in SEQ ID NO. 4, SEQ ID NO. 6 or SEQ ID NO. 8.
An isolated nucleic acid molecule comprising a polynucleotide sequence encoding a serine protease selected from the group consisting of: (a) A nucleic acid molecule encoding a polypeptide having serine protease activity and comprising an amino acid sequence as depicted in SEQ ID NO. 4 or SEQ ID NO. 6 or SEQ ID NO. 8; and (b) a nucleic acid molecule comprising a coding sequence of a nucleotide sequence as depicted in SEQ ID NO. 3 or SEQ ID NO. 5 or SEQ ID NO. 7 is also an aspect of the invention.
Another aspect of the invention is a recombinant expression vector comprising an isolated nucleic acid encoding a serine protease selected from the group consisting of: (a) A nucleic acid molecule encoding a polypeptide having serine protease activity and comprising an amino acid sequence as depicted in SEQ ID NO. 4 or SEQ ID NO. 6 or SEQ ID NO. 8; and (b) a nucleic acid molecule comprising a coding sequence of a nucleotide sequence as depicted in SEQ ID NO. 3 or SEQ ID NO. 5 or SEQ ID NO. 7, said nucleic acid being operably linked to a sequence capable of directing expression of said serine protease in a suitable host. Furthermore, in one aspect the invention provides an enzyme preparation comprising a recombinant serine protease. A preferred aspect of the invention is an enzyme preparation comprising a further enzyme with or without mediator selected from the group consisting of proteases, amylases, cellulases, lipases, xylanases, mannanases, cutinases, pectinases, polygalacturonases, pectin lyase, pectinolytic enzymes, esterases, phytases, arabinases, galactanases, huang Yuanmei, xyloglucanases, DNases and oxidases. Another preferred aspect of the invention is to include one or more additives selected from stabilizers, buffers, surfactants, builders, bleaching agents, media, anti-corrosion agents (anti-redeposition agents), corrosion agents (caustics), abrasives, optical brighteners, dyes, perfumes, pigments, and Preservatives (PRESERVATIVES). According to a preferred embodiment, the enzyme preparation is in the form of a liquid composition or a solid composition, for example in the form of a solution, dispersion, paste, powder, granule, granulate, coated granulate, tablet, cake, crystal slurry, gel or pill.
One aspect of the invention is a host cell comprising a recombinant expression vector of the invention. Preferably, the host is a microbial host. More preferably, the host is a filamentous fungus. Even more preferably, the host belongs to the genus Trichoderma, aspergillus, fusarium, humicola, chrysosporium, neurospora (Neurospora), rhizopus, penicillium, myceliophthora and Mortierella (Mortierella). Most preferably, the host is Trichoderma reesei.
One aspect of the present invention is a method for producing a protease, comprising the steps of: amino acid substitutions Q103D and/or S105E are introduced into a sequence comprising an amino acid sequence having at least 70% identity to the amino acid sequence defined in SEQ ID No. 2. A preferred aspect of the present invention is a method for producing a protease, wherein the method further comprises the steps of: culturing the host cell and recovering the enzyme.
According to a preferred embodiment, the recombinant serine protease or enzyme preparation of the invention can be used in detergents, in treating fibers, in treating proteinaceous material, in treating food or feed, or in applications involving modification, degradation or removal of proteinaceous material. A preferred embodiment is the use of the recombinant serine proteases or enzyme preparations of the invention as detergent additives. Another preferred embodiment is the use of the recombinant serine protease or enzyme preparation in a liquid detergent or a solid detergent, preferably in the form of a bar, a homogeneous tablet, a tablet with two or more layers, a bag with one or more compartments, a conventional or compact powder, granules, paste, gel, or a conventional, compact or concentrated liquid.
A detergent composition, characterized in that it comprises a surfactant, a recombinant serine protease or an enzyme preparation according to the invention, and an additive selected from the group consisting of stabilizers, buffers, builders, bleaching agents, media, corrosion inhibitors, anti-redeposition agents, corrosion inhibitors, abrasives, optical brighteners, dyes, perfumes, pigments and preservatives is also a preferred aspect of the invention. It is also a further aspect of the invention that the detergent composition comprises other enzymes with or without mediators selected from the group consisting of proteases, amylases, cellulases, lipases, xylanases, mannanases, cutinases, pectinases, polygalacturonases, pectate lyases, pectolyases, esterases, phytases, arabinanases, galactanases, huang Yuanmei, xyloglucanases, dnases and oxidases.
One aspect of the invention is a recombinant serine protease comprising an amino acid sequence having at least 70% identity to the amino acid sequence defined in SEQ ID NO. 2 and comprising amino acid D at position 103 and/or amino acid E at position 105 in the numbering according to SEQ ID NO. 2. A preferred aspect of the invention is a recombinant serine protease comprising an amino acid sequence having at least 70% identity to the amino acid sequence defined in SEQ ID NO. 2 and comprising amino acid D at position 103 and/or amino acid E at position 105 in the numbering according to SEQ ID NO. 2, wherein, in the amino acid sequence according to SEQ ID NO:2, said polypeptide having at least one amino acid substitution selected from the group consisting of Q103D and S105E. Still more preferred aspects of the invention are recombinant serine proteases comprising an amino acid sequence having at least 70% identity to the amino acid sequence defined in SEQ ID NO. 2 and comprising amino acid D at position 103 and/or amino acid E at position 105 in the numbering according to SEQ ID NO. 2, wherein the amino acid sequence thereof corresponds to the amino acid sequence of SEQ ID NO: 4. SEQ ID NO:6 or SEQ ID NO:8, and one of the amino acid sequences described in seq id no.
An isolated nucleic acid molecule comprising a polynucleotide sequence encoding a serine protease selected from the group consisting of:
(a) A nucleic acid molecule encoding a polypeptide having serine protease activity and comprising an amino acid sequence as depicted in SEQ ID NO. 4 or SEQ ID NO. 6 or SEQ ID NO. 8; and
(B) A nucleic acid molecule comprising a coding sequence of a nucleotide sequence as depicted in SEQ ID NO. 3 or SEQ ID NO. 5 or SEQ ID NO. 7.
Furthermore, a recombinant expression vector comprising an isolated nucleic acid comprising a polynucleotide sequence encoding a serine protease selected from the group consisting of: (a) A nucleic acid molecule encoding a polypeptide having serine protease activity and comprising an amino acid sequence as depicted in SEQ ID NO. 4 or SEQ ID NO. 6 or SEQ ID NO. 8; and (b) a nucleic acid molecule comprising a coding sequence of a nucleotide sequence as depicted in SEQ ID NO. 3 or SEQ ID NO. 5 or SEQ ID NO. 7, said nucleic acid being operably linked to regulatory sequences capable of directing the expression of said serine protease in a suitable host.
A host cell comprising a recombinant expression vector comprising an isolated nucleic acid comprising a polynucleotide sequence encoding a serine protease selected from the group consisting of: (a) A nucleic acid molecule encoding a polypeptide having serine protease activity and comprising the amino acid sequence depicted in SEQ ID NO. 4 or SEQ ID NO. 6 or SEQ ID NO. 8; and (b) a nucleic acid molecule comprising a coding sequence of a nucleotide sequence as depicted in SEQ ID NO. 3 or SEQ ID NO. 5 or SEQ ID NO. 7, said nucleic acid being operably linked to regulatory sequences capable of directing the expression of said serine protease in a suitable host. According to a more preferred aspect of the invention, the host cell is a microbial host cell. According to an even more preferred aspect of the invention, the host cell is a filamentous fungus. It is an even more preferred aspect of the invention that the host is Trichoderma, aspergillus, fusarium, humicola, chrysosporium, neurospora, rhizopus, penicillium, myceliophthora and Mortierella. According to a most preferred aspect of the invention, the host cell is Trichoderma reesei.
One aspect of the invention is a method for producing a protease comprising the step of introducing the amino acid substitutions Q103D and/or S105E into a sequence comprising an amino acid sequence having at least 70% identity to the amino acid sequence defined in SEQ ID No. 2.
According to a more preferred aspect, the method of producing a protease further comprises the step of culturing a host cell comprising a recombinant expression vector comprising an isolated nucleic acid comprising a polynucleotide sequence encoding a serine protease selected from the group consisting of: (a) A nucleic acid molecule encoding a polypeptide having serine protease activity and comprising the amino acid sequence depicted in SEQ ID NO. 4 or SEQ ID NO. 6 or SEQ ID NO. 8; and (b) a nucleic acid molecule comprising a coding sequence of a nucleotide sequence as depicted in SEQ ID NO. 3 or SEQ ID NO. 5 or SEQ ID NO. 7, said nucleic acid being operably linked to regulatory sequences capable of directing the expression of said serine protease in a suitable host.
An enzyme preparation comprising a recombinant serine protease comprising an amino acid sequence having at least 70% identity with the amino acid sequence defined in SEQ ID No. 2 and comprising amino acid D at position 103 and/or amino acid E at position 105 in the numbering according to SEQ ID No. 2. According to a more preferred aspect of the invention, the enzyme preparation comprises other enzymes with or without mediators selected from the group consisting of proteases, amylases, cellulases, lipases, xylanases, mannanases, cutinases, pectinases, polygalacturonases, pectin lyases, pectolyses, esterases, phytases, arabinases, galactanases, huang Yuanmei, xyloglucanases, dnases and oxidases.
According to an even more preferred aspect of the invention, the enzyme preparation comprises one or more additives selected from the group consisting of stabilizers, buffers, surfactants, builders, bleaching agents, media, corrosion inhibitors, anti-redeposition agents, corrosives, abrasives, optical brighteners, dyes, perfumes, pigments and preservatives. According to a most preferred aspect of the invention, the enzyme preparation is in the form of a liquid composition or a solid composition, for example in the form of a solution, dispersion, paste, powder, granule, granulate, coated granulate, tablet, cake, crystal slurry, gel or pill.
One aspect of the invention is the use of a recombinant serine protease or enzyme preparation comprising an amino acid sequence having at least 70% identity to the amino acid sequence defined in SEQ ID No. 2 and comprising amino acid D at position 103 and/or amino acid E at position 105 in the numbering according to SEQ ID No. 2, in an application for detergents, for treating fibres, for treating proteinaceous material, for treating food or feed, or for modification, degradation or removal involving proteinaceous material; the enzyme preparation comprises a recombinant serine protease comprising an amino acid sequence having at least 70% identity with the amino acid sequence defined in SEQ ID NO. 2 and comprising amino acid D at position 103 and/or amino acid E at position 105 in the numbering according to SEQ ID NO. 2.
According to a preferred aspect of the invention, the proteinaceous material is selected from the group consisting of wool, hair, leather and silk. According to an even more preferred aspect of the invention, the enzyme preparation is used as a detergent additive. According to a most preferred aspect of the invention, the use of the recombinant serine protease or enzyme preparation in a liquid detergent or a solid detergent, preferably a bar, a homogeneous tablet, a tablet with two or more layers, a bag with one or more compartments, a conventional or compact powder, a granule, a paste, a gel, or a conventional, compact or concentrated liquid.
Furthermore, a detergent composition is an aspect of the invention comprising a surfactant, a recombinant serine protease or an enzyme preparation, and an additive. The recombinant serine protease comprises an amino acid sequence having at least 70% identity to the amino acid sequence defined in SEQ ID NO. 2 and comprises amino acid D at position 103 and/or amino acid E at position 105 in the numbering according to SEQ ID NO. 2. The enzyme preparation comprises a recombinant serine protease comprising an amino acid sequence having at least 70% identity with the amino acid sequence defined in SEQ ID NO. 2 and comprising amino acid D at position 103 and/or amino acid E at position 105 in the numbering according to SEQ ID NO. 2. The additive is selected from the group consisting of stabilizers, buffers, builders, bleaching agents, media, corrosion inhibitors, anti-redeposition agents, corrosion inhibitors, abrasives, optical brighteners, dyes, perfumes, pigments, and preservatives.
Another aspect of the invention is a detergent composition comprising an additional enzyme with or without a mediator selected from the group consisting of proteases, amylases, cellulases, lipases, xylanases, mannanases, cutinases, pectinases, polygalacturonases, pectin lyase, pectinolytic enzymes, esterases, phytases, arabinanases, galactanases, huang Yuanmei, xyloglucanases, dnases, and oxidases.
As a conclusion, new fungal serine protease variants are provided which are derived from thermophilic microorganisms and are compatible and stable in liquid detergent compositions and less aggressive towards other enzymes, thus requiring less stabilizers and other additives.
The invention is illustrated by the following examples relating to some embodiments of the invention; however, the present invention is not meant to be limited to only these embodiments.
Examples
Example 1 design and production of a variant of a protease of the genus Butyrosporum in Trichoderma reesei
Based on the amino acid sequence of the mature wild-type arum ALK04122 protease, three arum protease variants were designed, designated M59 (Q103D), M60 (S105E) and M61 (Q103D; S105E) (Table 1), referred to herein as M0 (nucleic acid sequence encoding the amino acid sequence of mature arum ALK04122 protease SEQ ID NO:1, deduced amino acid sequence SEQ ID NO: 2). Variant genes were synthesized, each comprising the natural signal and nucleotide sequence of the pro sequence encoding the Butyrosporum ALK04122 protease (SEQ ID NO:9; FIG. 1), and the variant maturation coding sequence (see SEQ ID No. of Table 1) ordered from GenScript. The synthetic gene also includes, at its 5 '-and 3' -ends, trichoderma reesei sequences that are capable of correctly fusing with the cbhl promoter (SacII site is contained in the cbh1 promoter and the cbh1 promoter sequence from SacII to the promoter end) and the cbhl terminator (sequence after the cbh1 stop codon in the terminator to the AgeI site and AgeI site). There is Guan Lishi Trichoderma genomic sequences and gene organization, see https:// genome. Jgi. Doe. Gov/Trire 2.Home. Html. The synthetic gene was ligated into a SacII-AgeI digestion expression vector using standard molecular biology methods, the synthetic gene was cut from a commercial GenScript vector, and the expression cassette was constructed to produce recombinant protease variants (FIG. 2, table 1). In the final expression cassette, the synthetic gene encoding the full-length protease variant was fused (exact fusion) to the Trichoderma reesei cbhl (cel 7A) promoter and terminator, similar to the construction described in Paloheimo et al (2003). The synthetic amdS gene encoding Aspergillus nidulans amidase (AmdS) was used as a transformation marker in the expression cassette (FIG. 2).
TABLE 1 information on the design and use of synthetic genes for the construction of expression cassettes for the production of the arum protease variants in Trichoderma reesei. In the absence of signal and pro-sequence, the SEQ ID numbers encoding the mature protease of nucleotide sequence, mature amino acid sequence and mutated amino acid position correspond to the mature arum protease amino acid sequence (SEQ ID NO: 2). Two possible options for selecting the triplet codons for D and E, which are more commonly used by trichoderma reesei (GAC and GAT for D, GAA and GAG for E).
The expression cassettes pALK4521, pALK4522 and pALK4523 (fig. 2) were isolated from the vector backbone of the expression plasmid by NotI digestion and used to transform the trichoderma reesei host strain. Transformation of Trichoderma reesei protoplasts was performed according to Penttila et al (1987) using the modification described by Karhunen et al (1993). Transformants were selected on plates containing acetamide as the sole nitrogen source. One set of transformants from each transformation was purified by single conidia on selection plates and then sporulated on Potato Dextrose (PD) agar.
To produce the protease, transformants were inoculated from PD inclined planes into 50ml of complex lactosyl cellulase induction medium (Joutsjoki et al, 1993) buffered by 5% KH 2PO4 in 250ml Erlenmeyer flasks. The untransformed host strain and the previously constructed transformant producing the recombinant wild-type aryomyces protease (M0) were used as references in culture. After 5 days of culture at 30℃and 250rpm, the culture supernatant was analyzed for protease yield of the transformant.
All three mutant proteases were well produced by Trichoderma reesei. In SDS-PAGE gels, a major protein band of about 30kDa was detected from the spent culture supernatant, which corresponds to the expected molecular weight of the recombinant protease. Protease activity was analyzed using casein as substrate, as described in patent EP2691520B1, using the optimized absorbance range of this molecule 0,1-0,5 (target 0, 3-0,35). One protease unit (BPU) corresponds to the amount of enzyme activity which, under standard conditions, releases an amount of peptide fragment corresponding to 1 μg of tyrosine from casein in 1 minute. The corresponding protease activities were obtained from pALK4521, pALK4522 and pALK4523 transformants, as from the reference transformants producing the recombinant wild-type verticillium protease.
Trichoderma reesei transformants that produced optimal protease activity in shake flask culture were grown in laboratory scale bioreactors. Cellulase was used in the culture to induce the complex medium. The spent medium obtained from shake flasks or bioreactor cultures was used for application testing (examples 2-3).
Example 2 decontamination performance of an ALKO 4122 protease variant of the genus Pythium in liquid detergents
Shake flask culture samples of the asmyces protease variants produced in trichoderma (as described in example 1) were tested for their ability to remove protease sensitive standard stains with commercial liquid detergents at 40 ℃ and 16°dh water hardness and compared to the parent molecule M0. The following artificially soiled test cloths from the center of the test material BV (netherlands) were used: cocoa (E-112), blood/milk/ink, co+PES (El 17), blood/milk/ink, co (El 16), grass (El 64), whole egg with pigment (CS-37), egg yolk with pigment (CS-38), chocolate milk soot (C-03) and peanut oil, milk (C-10). The fabric was cut into 6 cm by 6 cm samples using a base of commercial liquid detergent without enzyme at a concentration of 5g per liter of wash liquor at a pH of about 8.2. Protease was administered in enzyme activity units (BPU) associated with μg tyrosine/min/ml wash liquor as described in example 1.
For synthetic tap water with hardness of 16°dh, the following stock solutions were prepared using deionized water (Milli-Q or equivalent):
Stock solution of 1000 ° d calcium hardness: caCl 2 x 2H2 O (1.02382.1000,Merck KGaA, germany) 26.22g/l
Stock solution of 200 ° d magnesium hardness: mgSO 4 x 7H2 O (1.05886.1000,Merck KGaA, germany) 8.79g/l H 2 O
NaHCO 3 stock solution: naHCO 3 (1.06329.1000Merck KGaA, germany) 29,6g/l
13,3Ml CaCl 2 solution, 13.3ml MgSO 4 solution and 10.0ml freshly prepared NaHCO 3 solution were added in the given order to a volumetric flask, and the flask was volumetric to 1 liter with deionized water and mixed. The hardness of the water was measured by complexometric titration and found to be correct.
The following decontamination treatments were performed in Atlas LP-2 primer-Ometer. The primer-Ometer was first preheated to 40 ℃. The detergent, 250ml of synthetic tap water with a hardness of 16°dh and diluted enzyme (< 1,0 ml) were then added to a 1.2 liter vessel. Stain was added and the primer-Ometer was run at 40℃for 60min at 42 rpm. The stains E-112, E-116, E-117 and E-164 are placed together in the same container, and the remaining stains are placed in another container. After this time, the samples were carefully rinsed under running water and dried overnight in room air on a grid protected from light.
The decontamination effect was evaluated by measuring color as reflectance values from the L x a x b x color space coordinates (light source D65/100, 420nm cut) using a Konica Minolta CM-3610A spectrophotometer. Stain fade was calculated as Δl (deltaL), which is the brightness value L of the enzyme treated fabric minus the brightness value L of the fabric treated with the protease-free wash liquor (control), indicating protease performance (stain removal efficiency). The final result (total stain removal) is shown as Δl sum for each stain.
The best detergent effect (AL sum of 8 stains) was obtained using shake flask supernatants of variants M60 and M61, as shown in fig. 3. The properties were surprisingly improved compared to the parent molecule M0, in particular on blood-milk-ink and egg stains (fig. 4 and 5).
M60 production-like samples (products LIKE SAMPLE of M60) from pilot cultures were also tested using a similar test system as described above but with a broader dosage range (0-12 BPU/ml wash), which represents the typical dosage range for commercial proteases (enzyme products of 0,1-1% detergent weight). The results of M0 production samples demonstrated that the stain removal performance of M60 was significantly better than that of the parent molecule M0 (fig. 6), especially blood-milk-ink (fig. 7A and 7B) and whole egg (fig. 7C) and egg yolk (fig. 7D) stains.
EXAMPLE 3 degradation of commercial cellulases in liquid detergents by protease variants at 37℃
Shake flask culture supernatants of the M0 variants produced in trichoderma as described in example 1 were tested based on their degradation of commercial cellulases in liquid detergents at 37 ℃. Parent molecule M0 was used as a reference. The protease was added in an amount corresponding to 520BPU/g activity to a commercially available cellulase containing 1% (w/w)DCL (by AB Enzymes) in liquid screen detergent. Protease activity was measured as described in example 1. The composition of the detergent is shown in Table 2. Samples in covered plastic tubes were incubated at 37℃for 7 days. Essentially as described in Bailey and Nevalainen,1981; haakana, 2004 (NCU Activity) the cellulase activity was measured as the released reducing sugar from carboxymethylcellulose (3% CMC) in 50mM HEPES buffer, pH7.0 at 50 ℃. The results were calculated as relative residual cellulase activity, which was obtained by dividing the residual activity of the sample containing M61 after incubation at 37 ℃ by the residual activity of the sample containing the parent molecule M0. Residual activity (%) was calculated by dividing the activity of the sample after incubation at 37 ℃ by the initial activity of the sample. In addition to cellulase activity (NCU), the residual protease activity (BPU) of the samples was also measured after 7 days using the method described in example 1.
Table 2. Composition of liquid detergent formulation.
Composition of the components ％
Anionic surfactants 10-20
Nonionic surfactant, soap 5-10
Boric acid ≤1
Propylene glycol 4-8％
Ethanol 2-5％
pH 8.0 -8.4
Based on the results shown in FIG. 8, the stability of the cellulase was improved without significant changes in protease activity in the presence of the variant protease in the liquid detergent at 37℃compared to the parent molecule M0 (data not shown). The best cellulase stability was obtained with shake flask culture samples of protease variants M60 and M61, which increased the stability of cellulase activity by about 1.4-fold when protease was added to the detergent at 520 BPU/g.
Stability testing was also performed on M60 production-like samples from pilot cultures using a similar test system as described above but with a large amount of protease corresponding to 2080BPU per gram of detergent. Parent molecule M0 was used as a reference.
The results obtained with M60 production samples demonstrated that there was still considerably more cellulase activity (about 1.8 fold) at high protease levels than the parent molecule M0 (FIG. 9) within 7 or 14 days, with no significant change in residual protease activity (data not shown). The results of these tests demonstrate that the variants of the invention have improved wash performance (detergency effect), and are less invasive to cellulases.
Reference to the literature
Anwar, A and M Saleumuldin 1998 alkaline protease: reviewed Bioresource Technology 64:175-183.
Bailey M and Nevalainen H.1981. Induction, isolation and testing of Stable Trichoderma reesei mutants with improved solubilised cellulase yields. Enzyme microb. Technology.3:153-1577.
Chen, Y-J and M Inouye, 2008. Intramolecular chaperone mediated protein folding Curr. Opin. Structure. Biol.18:765-770.
Directed evolution of industrial enzymes by Cherry, J.R. and Fidantsef, A.L.2003. Update. Curr. Opin. Biotechnol.14:438-443.
Gaucher G M, stevenson K J2004. Thermomycins, prohydrolase Manual 2 nd edition, 1834-1835.
Gupta, R, QK Beg, S Khan and B Chauhan.2002. Fermentation, downstream processing and characterization overview of microbial alkaline proteases. Appl. Microbiol. Biotechnol.60:381-395.
Haakana H,Miettinen-Oinonen A,Joutsjoki V,A, suominen P andJ.2004. cloning of the Ginkgo Biloba (Melanocarpus albomyce) cellulase Gene and efficient expression thereof in Trichoderma reesei Enzyme microb.technology.34:159-167.
Joutsjoki, V.V., T.K.Torkkeli, and K.M.H.Nevalainen.1993. Transformation of Trichoderma reesei with the Trichoderma resina glucoamylase P (gamP) gene Trichoderma reesei produced heterologous glucoamylase Curr.Genet.24:223-228.
Karhunen,T.,A.High frequency one-step gene substitution in overproduction of K.M.H.Nevalainen, and P.L.Suominen.1993. Trichoderma reesei I. Endoglucanase I. Mol.Gen.Genet.241:515-522.
Kalisz, HM.1988 microbial protease, adv.biochem.Eng.Biotechnol.36:1-65.
MALARDIER L, MJ Daboussi, J Julien, F Roussel, C Scazzocchio and Y Brygoo.1989. Cloning of Aspergillus nidulans nitrate reductase Gene (niaD) and its use in Fusarium oxysporum transformation. Gene 78:147-156.
Maurer, K-H.2004 detergent protease Curr. Opin. Biotechnol.15:330-334.
Maurer, K-H,2010. Enzymes, detergents, pp.1-17 (MC FLICKINGER ed.) industrial biotechnology encyclopedia: biological processes, bioseparation and cell technology, john Wiley & Sons, inc.
Ong PH and Gaucher GM,1975. Production, purification and characterization of the extracellular serine protease thermolysin of eukaryotic extracellular enzyme and thermomyces Malbranchea pulchella variant sulfurea. Can. J. Microbiol.22:165-175.
Paloheimo,M.,A.High production of bacterial xylanases in the filamentous fungus Trichoderma reesei requires a carrier polypeptide with an intact domain structure, appl. Env. Microbiol.69:7073-7082.
M,H Nevalainen,ME SALMINEN, and J knowles.1987. A multifunctional transformation system for the cellulolytic filamentous fungus Trichoderma reesei Gene 61:155-164.
Molecular and biotechnological aspects of Rao, MB, AM Tanksale, MS Ghatge and VV Deshpande.1998. Microprotease. Microbiol. Mol. Biol. Rev.62:597-635.
SEQUENCE LISTING
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<120> PROTEASE ENZYME VARIANTS AND USES THEREOF
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gcattggtga cgcagagtaa tgcaccatcc tggggccttg gccgtatttc caaccgacag 60
gctggtattc gtgattacca ctacgatgac tccgccggtg aaggcgtcat cgtctatgat 120
gttgacaccg gcattgacat cagccatccg gatttcgagg gccgtgctat atggggttcc 180
aaccatgtcg accgcgttaa ccaggatcag aatggccatg ggacacacgt tgctggtact 240
attggtggaa gggcgtacgg agtcgccaag aaggccacaa tagtggctgt caaggttctc 300
gacgcccagg ggtcaggtac tatcagcggt attattgctg gtcttgactg gagtgtcaat 360
catgctcgac agaatggagt cactagaaga gcggctttga acatgagcct tggcggtggg 420
cgcagtatct ctttcaatca ggctgctgca agtgctgtcc aagccggatt gttcgtcgcg 480
gttgctgccg gaaatgaagg ggtaagtgac ttctttctgg cccctcctat ccgtacctgc 540
agaagctaac cagattgctc ttattttttt tcttttttca aaatatagca aaatgcaggt 600
aacacttccc cagcctcaga gccttctgtt tgcacagtag gggcaacctc atcgaatgat 660
gccgccacat cctggtccaa ctatggctca gttggtacgt agggctcggt tttatttatt 720
acttcttccc cacatgcgat cagaccggcc gctgactata tttagttgac gtttacgctc 780
ccggagacgc aattgtctct acctggcccg gtggcggttc caggtctctc tcaggcacat 840
cgatggcttc tccacacgtc gccggcctgg gtgcatacct catcgctctg gagggcatta 900
gcggaggcag tgtatgtgac cgtatcaaag agctggctca acctgtcgtc cagcctggtc 960
caggcaccac caaccgtctt atctacaacg gcagtggccg ctaa 1004
<210> 2
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<223> mature protease amino acid sequence
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Ala Leu Val Thr Gln Ser Asn Ala Pro Ser Trp Gly Leu Gly Arg Ile
1 5 10 15
Ser Asn Arg Gln Ala Gly Ile Arg Asp Tyr His Tyr Asp Asp Ser Ala
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Gly Glu Gly Val Ile Val Tyr Asp Val Asp Thr Gly Ile Asp Ile Ser
35 40 45
His Pro Asp Phe Glu Gly Arg Ala Ile Trp Gly Ser Asn His Val Asp
50 55 60
Arg Val Asn Gln Asp Gln Asn Gly His Gly Thr His Val Ala Gly Thr
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Ile Gly Gly Arg Ala Tyr Gly Val Ala Lys Lys Ala Thr Ile Val Ala
85 90 95
Val Lys Val Leu Asp Ala Gln Gly Ser Gly Thr Ile Ser Gly Ile Ile
100 105 110
Ala Gly Leu Asp Trp Ser Val Asn His Ala Arg Gln Asn Gly Val Thr
115 120 125
Arg Arg Ala Ala Leu Asn Met Ser Leu Gly Gly Gly Arg Ser Ile Ser
130 135 140
Phe Asn Gln Ala Ala Ala Ser Ala Val Gln Ala Gly Leu Phe Val Ala
145 150 155 160
Val Ala Ala Gly Asn Glu Gly Gln Asn Ala Gly Asn Thr Ser Pro Ala
165 170 175
Ser Glu Pro Ser Val Cys Thr Val Gly Ala Thr Ser Ser Asn Asp Ala
180 185 190
Ala Thr Ser Trp Ser Asn Tyr Gly Ser Val Val Asp Val Tyr Ala Pro
195 200 205
Gly Asp Ala Ile Val Ser Thr Trp Pro Gly Gly Gly Ser Arg Ser Leu
210 215 220
Ser Gly Thr Ser Met Ala Ser Pro His Val Ala Gly Leu Gly Ala Tyr
225 230 235 240
Leu Ile Ala Leu Glu Gly Ile Ser Gly Gly Ser Val Cys Asp Arg Ile
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Lys Glu Leu Ala Gln Pro Val Val Gln Pro Gly Pro Gly Thr Thr Asn
260 265 270
Arg Leu Ile Tyr Asn Gly Ser Gly Arg
275 280
<210> 3
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<213> Malbranchea cinnamomea
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<223> M59 mature nucleotide sequence
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gcattggtga cgcagagtaa tgcaccatcc tggggccttg gccgtatttc caaccgacag 60
gctggtattc gtgattacca ctacgatgac tccgccggtg aaggcgtcat cgtctatgat 120
gttgacaccg gcattgacat cagccatccg gatttcgagg gccgtgctat atggggttcc 180
aaccatgtcg accgcgttaa ccaggatcag aatggccatg ggacacacgt tgctggtact 240
attggtggaa gggcgtacgg agtcgccaag aaggccacaa tagtggctgt caaggttctc 300
gacgccgarg ggtcaggtac tatcagcggt attattgctg gtcttgactg gagtgtcaat 360
catgctcgac agaatggagt cactagaaga gcggctttga acatgagcct tggcggtggg 420
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gttgctgccg gaaatgaagg ggtaagtgac ttctttctgg cccctcctat ccgtacctgc 540
agaagctaac cagattgctc ttattttttt tcttttttca aaatatagca aaatgcaggt 600
aacacttccc cagcctcaga gccttctgtt tgcacagtag gggcaacctc atcgaatgat 660
gccgccacat cctggtccaa ctatggctca gttggtacgt agggctcggt tttatttatt 720
acttcttccc cacatgcgat cagaccggcc gctgactata tttagttgac gtttacgctc 780
ccggagacgc aattgtctct acctggcccg gtggcggttc caggtctctc tcaggcacat 840
cgatggcttc tccacacgtc gccggcctgg gtgcatacct catcgctctg gagggcatta 900
gcggaggcag tgtatgtgac cgtatcaaag agctggctca acctgtcgtc cagcctggtc 960
caggcaccac caaccgtctt atctacaacg gcagtggccg ctaa 1004
<210> 4
<211> 281
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<213> Malbranchea cinnamomea
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<223> M59 mature amino acid sequence
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Ala Leu Val Thr Gln Ser Asn Ala Pro Ser Trp Gly Leu Gly Arg Ile
1 5 10 15
Ser Asn Arg Gln Ala Gly Ile Arg Asp Tyr His Tyr Asp Asp Ser Ala
20 25 30
Gly Glu Gly Val Ile Val Tyr Asp Val Asp Thr Gly Ile Asp Ile Ser
35 40 45
His Pro Asp Phe Glu Gly Arg Ala Ile Trp Gly Ser Asn His Val Asp
50 55 60
Arg Val Asn Gln Asp Gln Asn Gly His Gly Thr His Val Ala Gly Thr
65 70 75 80
Ile Gly Gly Arg Ala Tyr Gly Val Ala Lys Lys Ala Thr Ile Val Ala
85 90 95
Val Lys Val Leu Asp Ala Asp Gly Ser Gly Thr Ile Ser Gly Ile Ile
100 105 110
Ala Gly Leu Asp Trp Ser Val Asn His Ala Arg Gln Asn Gly Val Thr
115 120 125
Arg Arg Ala Ala Leu Asn Met Ser Leu Gly Gly Gly Arg Ser Ile Ser
130 135 140
Phe Asn Gln Ala Ala Ala Ser Ala Val Gln Ala Gly Leu Phe Val Ala
145 150 155 160
Val Ala Ala Gly Asn Glu Gly Gln Asn Ala Gly Asn Thr Ser Pro Ala
165 170 175
Ser Glu Pro Ser Val Cys Thr Val Gly Ala Thr Ser Ser Asn Asp Ala
180 185 190
Ala Thr Ser Trp Ser Asn Tyr Gly Ser Val Val Asp Val Tyr Ala Pro
195 200 205
Gly Asp Ala Ile Val Ser Thr Trp Pro Gly Gly Gly Ser Arg Ser Leu
210 215 220
Ser Gly Thr Ser Met Ala Ser Pro His Val Ala Gly Leu Gly Ala Tyr
225 230 235 240
Leu Ile Ala Leu Glu Gly Ile Ser Gly Gly Ser Val Cys Asp Arg Ile
245 250 255
Lys Glu Leu Ala Gln Pro Val Val Gln Pro Gly Pro Gly Thr Thr Asn
260 265 270
Arg Leu Ile Tyr Asn Gly Ser Gly Arg
275 280
<210> 5
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gcattggtga cgcagagtaa tgcaccatcc tggggccttg gccgtatttc caaccgacag 60
gctggtattc gtgattacca ctacgatgac tccgccggtg aaggcgtcat cgtctatgat 120
gttgacaccg gcattgacat cagccatccg gatttcgagg gccgtgctat atggggttcc 180
aaccatgtcg accgcgttaa ccaggatcag aatggccatg ggacacacgt tgctggtact 240
attggtggaa gggcgtacgg agtcgccaag aaggccacaa tagtggctgt caaggttctc 300
gacgcccagg gggayggtac tatcagcggt attattgctg gtcttgactg gagtgtcaat 360
catgctcgac agaatggagt cactagaaga gcggctttga acatgagcct tggcggtggg 420
cgcagtatct ctttcaatca ggctgctgca agtgctgtcc aagccggatt gttcgtcgcg 480
gttgctgccg gaaatgaagg ggtaagtgac ttctttctgg cccctcctat ccgtacctgc 540
agaagctaac cagattgctc ttattttttt tcttttttca aaatatagca aaatgcaggt 600
aacacttccc cagcctcaga gccttctgtt tgcacagtag gggcaacctc atcgaatgat 660
gccgccacat cctggtccaa ctatggctca gttggtacgt agggctcggt tttatttatt 720
acttcttccc cacatgcgat cagaccggcc gctgactata tttagttgac gtttacgctc 780
ccggagacgc aattgtctct acctggcccg gtggcggttc caggtctctc tcaggcacat 840
cgatggcttc tccacacgtc gccggcctgg gtgcatacct catcgctctg gagggcatta 900
gcggaggcag tgtatgtgac cgtatcaaag agctggctca acctgtcgtc cagcctggtc 960
caggcaccac caaccgtctt atctacaacg gcagtggccg ctaa 1004
<210> 6
<211> 281
<212> PRT
<213> Malbranchea cinnamomea
<220>
<223> M60 mature amino acid sequence
<400> 6
Ala Leu Val Thr Gln Ser Asn Ala Pro Ser Trp Gly Leu Gly Arg Ile
1 5 10 15
Ser Asn Arg Gln Ala Gly Ile Arg Asp Tyr His Tyr Asp Asp Ser Ala
20 25 30
Gly Glu Gly Val Ile Val Tyr Asp Val Asp Thr Gly Ile Asp Ile Ser
35 40 45
His Pro Asp Phe Glu Gly Arg Ala Ile Trp Gly Ser Asn His Val Asp
50 55 60
Arg Val Asn Gln Asp Gln Asn Gly His Gly Thr His Val Ala Gly Thr
65 70 75 80
Ile Gly Gly Arg Ala Tyr Gly Val Ala Lys Lys Ala Thr Ile Val Ala
85 90 95
Val Lys Val Leu Asp Ala Gln Gly Glu Gly Thr Ile Ser Gly Ile Ile
100 105 110
Ala Gly Leu Asp Trp Ser Val Asn His Ala Arg Gln Asn Gly Val Thr
115 120 125
Arg Arg Ala Ala Leu Asn Met Ser Leu Gly Gly Gly Arg Ser Ile Ser
130 135 140
Phe Asn Gln Ala Ala Ala Ser Ala Val Gln Ala Gly Leu Phe Val Ala
145 150 155 160
Val Ala Ala Gly Asn Glu Gly Gln Asn Ala Gly Asn Thr Ser Pro Ala
165 170 175
Ser Glu Pro Ser Val Cys Thr Val Gly Ala Thr Ser Ser Asn Asp Ala
180 185 190
Ala Thr Ser Trp Ser Asn Tyr Gly Ser Val Val Asp Val Tyr Ala Pro
195 200 205
Gly Asp Ala Ile Val Ser Thr Trp Pro Gly Gly Gly Ser Arg Ser Leu
210 215 220
Ser Gly Thr Ser Met Ala Ser Pro His Val Ala Gly Leu Gly Ala Tyr
225 230 235 240
Leu Ile Ala Leu Glu Gly Ile Ser Gly Gly Ser Val Cys Asp Arg Ile
245 250 255
Lys Glu Leu Ala Gln Pro Val Val Gln Pro Gly Pro Gly Thr Thr Asn
260 265 270
Arg Leu Ile Tyr Asn Gly Ser Gly Arg
275 280
<210> 7
<211> 1004
<212> DNA
<213> Malbranchea cinnamomea
<220>
<223> M61 mature nucleotide sequence
<220>
<221> misc_feature
<222> (309)..(309)
<223> r is t or c
<220>
<221> misc_feature
<222> (315)..(315)
<223> y is g or a
<400> 7
gcattggtga cgcagagtaa tgcaccatcc tggggccttg gccgtatttc caaccgacag 60
gctggtattc gtgattacca ctacgatgac tccgccggtg aaggcgtcat cgtctatgat 120
gttgacaccg gcattgacat cagccatccg gatttcgagg gccgtgctat atggggttcc 180
aaccatgtcg accgcgttaa ccaggatcag aatggccatg ggacacacgt tgctggtact 240
attggtggaa gggcgtacgg agtcgccaag aaggccacaa tagtggctgt caaggttctc 300
gacgccgarg gggayggtac tatcagcggt attattgctg gtcttgactg gagtgtcaat 360
catgctcgac agaatggagt cactagaaga gcggctttga acatgagcct tggcggtggg 420
cgcagtatct ctttcaatca ggctgctgca agtgctgtcc aagccggatt gttcgtcgcg 480
gttgctgccg gaaatgaagg ggtaagtgac ttctttctgg cccctcctat ccgtacctgc 540
agaagctaac cagattgctc ttattttttt tcttttttca aaatatagca aaatgcaggt 600
aacacttccc cagcctcaga gccttctgtt tgcacagtag gggcaacctc atcgaatgat 660
gccgccacat cctggtccaa ctatggctca gttggtacgt agggctcggt tttatttatt 720
acttcttccc cacatgcgat cagaccggcc gctgactata tttagttgac gtttacgctc 780
ccggagacgc aattgtctct acctggcccg gtggcggttc caggtctctc tcaggcacat 840
cgatggcttc tccacacgtc gccggcctgg gtgcatacct catcgctctg gagggcatta 900
gcggaggcag tgtatgtgac cgtatcaaag agctggctca acctgtcgtc cagcctggtc 960
caggcaccac caaccgtctt atctacaacg gcagtggccg ctaa 1004
<210> 8
<211> 281
<212> PRT
<213> Malbranchea cinnamomea
<220>
<223> M61 mature amino acid sequence
<400> 8
Ala Leu Val Thr Gln Ser Asn Ala Pro Ser Trp Gly Leu Gly Arg Ile
1 5 10 15
Ser Asn Arg Gln Ala Gly Ile Arg Asp Tyr His Tyr Asp Asp Ser Ala
20 25 30
Gly Glu Gly Val Ile Val Tyr Asp Val Asp Thr Gly Ile Asp Ile Ser
35 40 45
His Pro Asp Phe Glu Gly Arg Ala Ile Trp Gly Ser Asn His Val Asp
50 55 60
Arg Val Asn Gln Asp Gln Asn Gly His Gly Thr His Val Ala Gly Thr
65 70 75 80
Ile Gly Gly Arg Ala Tyr Gly Val Ala Lys Lys Ala Thr Ile Val Ala
85 90 95
Val Lys Val Leu Asp Ala Asp Gly Glu Gly Thr Ile Ser Gly Ile Ile
100 105 110
Ala Gly Leu Asp Trp Ser Val Asn His Ala Arg Gln Asn Gly Val Thr
115 120 125
Arg Arg Ala Ala Leu Asn Met Ser Leu Gly Gly Gly Arg Ser Ile Ser
130 135 140
Phe Asn Gln Ala Ala Ala Ser Ala Val Gln Ala Gly Leu Phe Val Ala
145 150 155 160
Val Ala Ala Gly Asn Glu Gly Gln Asn Ala Gly Asn Thr Ser Pro Ala
165 170 175
Ser Glu Pro Ser Val Cys Thr Val Gly Ala Thr Ser Ser Asn Asp Ala
180 185 190
Ala Thr Ser Trp Ser Asn Tyr Gly Ser Val Val Asp Val Tyr Ala Pro
195 200 205
Gly Asp Ala Ile Val Ser Thr Trp Pro Gly Gly Gly Ser Arg Ser Leu
210 215 220
Ser Gly Thr Ser Met Ala Ser Pro His Val Ala Gly Leu Gly Ala Tyr
225 230 235 240
Leu Ile Ala Leu Glu Gly Ile Ser Gly Gly Ser Val Cys Asp Arg Ile
245 250 255
Lys Glu Leu Ala Gln Pro Val Val Gln Pro Gly Pro Gly Thr Thr Asn
260 265 270
Arg Leu Ile Tyr Asn Gly Ser Gly Arg
275 280
<210> 9
<211> 1436
<212> DNA
<213> Malbranchea cinnamomea
<220>
<223> The nucleotide sequence encoding the full-length amino acid
sequence of Malbranchea ALKO4122 protease. The full-length gene
in included in plasmid pALK3094. The protease sequence cloned
from Malbranchea ALKO4178 by PCR was identical to this sequence.
<400> 9
atgggcgtct tcagcaaact cttgtatctg tcttttgcag tcacggcctc tgtcaatgcc 60
ggtgaaatcc tttcagtcgc caacaaggac agtgttatcc ctgacacgta tatcgtggtg 120
ttgaaggaag gagtttcaac ccaggagttc aatgctcata aaaactgggt gaacgagatt 180
catcgcacca acctcacgag gcgtgacctg ggtttcactg gcgagttaaa gcatagctat 240
gattttggtg gacatggact gaagggctac agcggcaagt ttgatgccac tgccattcag 300
gaaattgcca atgatcctaa tgtatgcttg ttaagaattc ttcccagcga gatatcttca 360
tgcaagccat gcaattgctg acaggtgaat taggtggcct acgtcgaacc ggaccaggag 420
gtgaagcttg atgcattggt gacgcagagt aatgcaccat cctggggcct tggccgtatt 480
tccaaccgac aggctggtat tcgtgattac cactacgatg actccgccgg tgaaggcgtc 540
atcgtctatg atgttgacac cggtattgac atcagccatc cggatttcga gggccgtgct 600
atatggggtt ccaaccatgt cgaccgcgtt aaccaggatc agaatggcca tgggacacac 660
gttgctggta ctattggtgg aagggcgtac ggagtcgcca agaaggccac aatagtggct 720
gtcaaggttc tcgacgccca ggggtcaggt actatcagcg gtattattgc tggtcttgac 780
tggagtgtca atcatgctcg acagaatgga gtcactagaa gagcggcttt gaacatgagc 840
cttggcggtg ggcgcagtat ctctttcaat caggctgctg caagtgctgt ccaagccgga 900
ttgttcgtcg cggttgctgc cggaaatgaa ggggtaagtg acttctttct ggcccctcct 960
atccgtacct gcagaagcta accagattgc tcttattttt tttctttttt caaaatatag 1020
caaaatgcag gtaacacttc cccagcctca gagccttctg tttgcacagt aggggcaacc 1080
tcatcgaatg atgccgccac atcctggtcc aactatggct cagttggtac gtagggctcg 1140
gttttattta ttacttcttc cccacatgcg atcagaccgg ccgctgacta tatttagttg 1200
acgtttacgc tcccggagac gcaattgtct ctacctggcc cggtggcggt tccaggtctc 1260
tctcaggcac atcgatggct tctccacacg tcgccggcct gggtgcatac ctcatcgctc 1320
tggagggcat tagcggaggc agtgtatgtg accgtatcaa agagctggct caacctgtcg 1380
tccagcctgg tccaggcacc accaaccgtc ttatctacaa cggcagtggc cgctaa 1436
<210> 10
<211> 401
<212> PRT
<213> Malbranchea cinnamomea
<220>
<223> The full-length amino acid sequence of Malbranchea ALKO4122
protease including amino acids Met1 to Arg 401 of the full length
protease.
<400> 10
Met Gly Val Phe Ser Lys Leu Leu Tyr Leu Ser Phe Ala Val Thr Ala
1 5 10 15
Ser Val Asn Ala Gly Glu Ile Leu Ser Val Ala Asn Lys Asp Ser Val
20 25 30
Ile Pro Asp Thr Tyr Ile Val Val Leu Lys Glu Gly Val Ser Thr Gln
35 40 45
Glu Phe Asn Ala His Lys Asn Trp Val Asn Glu Ile His Arg Thr Asn
50 55 60
Leu Thr Arg Arg Asp Leu Gly Phe Thr Gly Glu Leu Lys His Ser Tyr
65 70 75 80
Asp Phe Gly Gly His Gly Leu Lys Gly Tyr Ser Gly Lys Phe Asp Ala
85 90 95
Thr Ala Ile Gln Glu Ile Ala Asn Asp Pro Asn Val Ala Tyr Val Glu
100 105 110
Pro Asp Gln Glu Val Lys Leu Asp Ala Leu Val Thr Gln Ser Asn Ala
115 120 125
Pro Ser Trp Gly Leu Gly Arg Ile Ser Asn Arg Gln Ala Gly Ile Arg
130 135 140
Asp Tyr His Tyr Asp Asp Ser Ala Gly Glu Gly Val Ile Val Tyr Asp
145 150 155 160
Val Asp Thr Gly Ile Asp Ile Ser His Pro Asp Phe Glu Gly Arg Ala
165 170 175
Ile Trp Gly Ser Asn His Val Asp Arg Val Asn Gln Asp Gln Asn Gly
180 185 190
His Gly Thr His Val Ala Gly Thr Ile Gly Gly Arg Ala Tyr Gly Val
195 200 205
Ala Lys Lys Ala Thr Ile Val Ala Val Lys Val Leu Asp Ala Gln Gly
210 215 220
Ser Gly Thr Ile Ser Gly Ile Ile Ala Gly Leu Asp Trp Ser Val Asn
225 230 235 240
His Ala Arg Gln Asn Gly Val Thr Arg Arg Ala Ala Leu Asn Met Ser
245 250 255
Leu Gly Gly Gly Arg Ser Ile Ser Phe Asn Gln Ala Ala Ala Ser Ala
260 265 270
Val Gln Ala Gly Leu Phe Val Ala Val Ala Ala Gly Asn Glu Gly Gln
275 280 285
Asn Ala Gly Asn Thr Ser Pro Ala Ser Glu Pro Ser Val Cys Thr Val
290 295 300
Gly Ala Thr Ser Ser Asn Asp Ala Ala Thr Ser Trp Ser Asn Tyr Gly
305 310 315 320
Ser Val Val Asp Val Tyr Ala Pro Gly Asp Ala Ile Val Ser Thr Trp
325 330 335
Pro Gly Gly Gly Ser Arg Ser Leu Ser Gly Thr Ser Met Ala Ser Pro
340 345 350
His Val Ala Gly Leu Gly Ala Tyr Leu Ile Ala Leu Glu Gly Ile Ser
355 360 365
Gly Gly Ser Val Cys Asp Arg Ile Lys Glu Leu Ala Gln Pro Val Val
370 375 380
Gln Pro Gly Pro Gly Thr Thr Asn Arg Leu Ile Tyr Asn Gly Ser Gly
385 390 395 400
Arg

Claims (17)

1. A recombinant serine protease having an amino acid sequence as set forth in SEQ ID NO. 4, SEQ ID NO. 6 or SEQ ID NO. 8.
2. An isolated nucleic acid molecule comprising a polynucleotide sequence encoding a serine protease selected from the group consisting of:
(a) A nucleic acid molecule encoding a polypeptide having serine protease activity and having the amino acid sequence depicted in SEQ ID NO. 4 or SEQ ID NO. 6 or SEQ ID NO. 8; and
(B) The coding sequence of the nucleotide sequence is the nucleic acid molecule described in SEQ ID NO. 3 or SEQ ID NO. 5 or SEQ ID NO. 7.
3. A recombinant expression vector comprising the isolated nucleic acid molecule of claim 2 operably linked to regulatory sequences capable of directing expression of the serine protease in a suitable host.
4. A host cell comprising the recombinant expression vector of claim 3.
5. The host cell of claim 4, wherein the host is trichoderma reesei.
6. A method of producing a protease, the method comprising the steps of: producing a variant of a protease of the genus Butyrosporum in Trichoderma reesei, said variant having an amino acid sequence of one of the amino acid sequences depicted in SEQ ID NO. 4, SEQ ID NO. 6 or SEQ ID NO. 8.
7. The method for producing a protease according to claim 6, further comprising the step of: culturing the host cell of claim 4 or 5 and recovering the enzyme.
8. An enzyme preparation comprising the recombinant serine protease of claim 1.
9. The enzyme preparation according to claim 8, characterized in that the preparation comprises other enzymes selected from the group consisting of proteases, amylases, cellulases, lipases, xylanases, mannanases, cutinases, pectinases, polygalacturonases, esterases, phytases, arabinases, galactanases, huang Yuanmei, xyloglucanases, dnases and oxidases, with or without mediators.
10. The enzyme preparation according to claim 8 or 9, characterized in that the preparation comprises one or more additives selected from the group consisting of stabilizers, buffers, surfactants, builders, bleaching agents, antiredeposition agents, corrosives, abrasives, optical brighteners, dyes, perfumes, pigments and preservatives.
11. The enzyme preparation according to claim 8 or 9, characterized in that the enzyme preparation is in the form of a liquid composition or a solid composition selected from the group consisting of solutions, dispersions, pastes, powders, granules, granulates, coated granulates, tablets, cakes, crystals, crystal slurries, gels or pills.
12. Use of a recombinant serine protease according to claim 1 or an enzyme preparation according to any one of claims 8-11 in applications for detergents, for treating fibres, for treating proteinaceous material, for treating food or feed or for modification, degradation or removal involving proteinaceous material.
13. Use according to claim 12, wherein the protein material is selected from wool, hair, leather and silk.
14. Use of the recombinant serine protease of claim 1 or the enzyme preparation of any one of claims 8-11 as a detergent additive.
15. Use of the recombinant serine protease of claim 1 or the enzyme preparation of any one of claims 8-11 in a liquid detergent or a solid detergent in the form of a bar, a homogeneous tablet, a tablet with two or more layers, a bag with one or more compartments, a conventional or compact powder, a granule, a paste, a gel or a conventional, compact or concentrated liquid.
16. A detergent composition, said composition comprising: (a) a surfactant, (b) a recombinant serine protease according to claim 1 or an enzyme preparation according to any one of claims 8-11, and (c) an additive selected from the group consisting of stabilizers, buffers, builders, bleaches, anti-redeposition agents, corrosives, abrasives, optical brighteners, dyes, perfumes, pigments and preservatives.
17. The detergent composition according to claim 16, characterized in that the composition comprises other enzymes selected from proteases, amylases, cellulases, lipases, xylanases, mannanases, cutinases, pectinases, polygalacturonases, esterases, phytases, arabinanases, galactanases, huang Yuanmei, xyloglucanases, dnases and oxidases, with or without mediators.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102597240A (en) * 2009-04-30 2012-07-18 生化酶股份有限公司 A fungal serine protease and use thereof
CN102625810A (en) * 2009-07-08 2012-08-01 生化酶股份有限公司 A fungal protease and use thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102597240A (en) * 2009-04-30 2012-07-18 生化酶股份有限公司 A fungal serine protease and use thereof
CN102625810A (en) * 2009-07-08 2012-08-01 生化酶股份有限公司 A fungal protease and use thereof

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